Antisense oligonucleotide compositions and methods for the modulation of activating protein 1

ABSTRACT

Compositions and methods for the treatment and diagnosis of diseases or disorders amenable to treatment through modulation of Activating Protein 1 (AP-1) expression are provided. In accordance with various embodiments of the present invention, oligonucleotides are provided which are specifically hybridizable with c-fos or c-jun, the genes encoding c-Fos or c-Jun, respectively. In a preferred embodiment, a method of modulating the metastasis of malignant tumors via modulation of one or more of the AP-1 subunits is provided; this method can be effected using the oligonucleotides of the invention or any other agent which modulates AP-1 or AP-1-mediated transcription.

This application is a continuation of Ser. No. 08/837,201, filed Apr.14, 1997.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatinglevels of the c-fos and c-jun genes, which encode the c-Fos and c-Junsubunits of AP-1, respectively. In vivo, AP-1, or transcription factoractivating protein 1, is a heterogenous mixture of heterodimers ofseveral related protein subunits including, in addition to c-Fos andc-Jun, FosB, Fra-1, Fra-2, c-Jun, JunB, JunD, etc. (The FOS and JUNFamilies of Proteins, Angel and Herrlich, eds., CRC Press, Boca Raton,Fla., 1994). AP-1 has been implicated in abnormal cell proliferation andtumor formation, events that thus might be controlled by modulating theexpression of c-fos and/or c-jun. The invention is further directed totherapeutic, diagnostic, and research based reagents and methods forevaluating and treating disease states or disorders which result fromand/or respond positively to modulation of one or more AP-1 subunits.Such disease states and disorders include those involving thehyperproliferation of cells such as, e.g., a tumor (neoplasm) ormalignant cancer. Inhibition of AP-1-mediated hyperproliferation ofcells, and corresponding prophylactic, palliative and therapeuticeffects result from treatment with the oligonucleotides of theinvention.

BACKGROUND OF THE INVENTION

Transcription factors play a central role in the expression of specificgenes upon stimulation by extracellular signals, thereby regulating acomplex array of biological processes. Members of the family oftranscription factors termed AP-1 (activating protein-1) alter geneexpression in response to growth factors, cytokines, tumor promoters,carcinogens and increased expression of certain oncogenes. Growthfactors and cytokines exert their function by binding to specific cellsurface receptors. Receptor occupancy triggers a signal transductioncascade to the nucleus. In this cascade, transcription factors such asAP-1 execute long term responses to the extracellular factors bymodulating gene expression. Such changes in cellular gene expressionlead to DNA synthesis, and eventually the formation of differentiatedderivatives (Angel and Karin, Biochim. Biophys. Acta, 1991, 1072, 129).

In general terms, AP-1 denotes one member of a family of relatedheterodimeric transcription factor complexes found in eukaryotic cellsor viruses. However, as used herein, “AP-1” specifically refers to theheterodimer formed of c-Fos and c-Jun (Angel and Herrlich, Chapter 1,and Schuermann, Chapter 2 in: The FOS and JUN Families of Proteins,Angel and Herrlich, eds., pp. 3-35, CRC Press, Boca Raton, Fla., 1994;Bohmann et al., Science, 1987, 238, 1386; Angel et al., Nature, 1988,332, 166). These two proteins are products of the c-fos and c-junproto-oncogenes, respectively. Repression of expression of either c-fosor c-jun, or of both proto-oncogenes, and the resultant inhibition ofthe formation of c-Fos and c-Jun proteins, is desirable for theinhibition of cell proliferation, tumor formation and tumor growth.

The phosphorylation of proteins plays a key role in the transduction ofextracellular signals into the cell. Mitogen-activated protein (MAP)kinases, enzymes which effect such phosphorylations are targets for theaction of growth factors, hormones, and other agents involved incellular metabolism, proliferation and differentiation (Cobb et al., J.Biol. Chem., 1995, 270, 14843). MAP kinases are themselves activated byphosphorylation catalyzed by, e.g., receptor tyrosine kinases, Gprotein-coupled receptors, protein kinase C (PKC), and the apparentlyMAP kinase dedicated kinases MEK1 and MEK2. MAP kinases include, but arenot limited to, ERK1, ERK2, two isoforms of ERK3, ERK4 (ERK stands for“extracellular signal-regulated protein kinase), Jun N-terminalkinases/stress-activated protein kinases (JNKs/SAPKs), p38/HOG1, p57 MAPkinases, MKK3 (MAP kinase kinase 3) and MKK4 (MAP kinase kinase 4, alsoknown as SAPK/ERK kinase (SEK) or JNK kinase (JNKK)) (Cobb et al., J.Biol. Chem., 1995, 270, 14843, and references cited therein). Ingeneral, MAP kinases are involved in a variety of signal transductionpathways (sometimes overlapping and sometimes parallel) that function toconvey extracellular stimuli to protooncogene products to modulatecellular proliferation and/or differentiation.

One of the signal transduction pathways involves the MAP kinases Junkinase 1 and Jun kinase 2 which are responsible for the phosphorylationof specific sites (Serine 63 and Serine 73) on c-Jun. Phosphorylation ofthese sites potentiates the ability of AP-1 to activate transcription(Binetruy et al., Nature, 1991, 351, 122; Smeal et al., Nature, 1991,354, 494). At least one human leukemia oncogene has been shown toenhance Jun N-terminal Kinase (JNK) function (Raitano et al., Proc.Natl. Acad. Sci. (USA), 1995, 92, 11746), thus indirectly demonstratinga role for AP-1 in cellular hyperproliferation and tumorigenesis.Cellular hyperproliferation in an animal can have several outcomes.Hyperproliferating cells might be attacked and killed by the animal'simmune system before a tumor can form., Tumors are abnormal growthsresulting from the hyperproliferation of cells. Cells that proliferateto excess but stay put form benign tumors, which can typically beremoved by local surgery. In contrast, malignant tumors or cancerscomprise cells that are capable of undergoing metastasis, i.e., aprocess by which hyperproliferative cells spread to, and securethemselves within, other parts of the body via the circulatory orlymphatic system (see, generally, Chapter 16 In: Molecular Biology ofthe Cell, Alberts et al., eds., pp. 891-950, Garland Publishing, Inc.,New York, 1983). Using the oligonucleotides of the invention, it hassurprisingly been discovered that several genes encoding enzymesrequired for metastasis are positively regulated by AP-1. Accordingly,inhibition of expression of c-fos and/or c-jun serves as a means tomodulate the metastasis of malignant tumors. A method of modulating oneor more metastatic events using the oligonucleotides of the invention isthus herein provided.

RELEVANT ART

Soprano et al. (Ann. N. Y. Acad. Sci., 1992, 660, 231) have usedantisense oligodeoxynucleotides targeted to c-jun mRNA to study theirability to inhibit DNA synthesis and cell division.

Liu et al. (Ann. Neurol., 1994, 36, 566) describe the suppression ofc-fos by intraventricular infusion of an antisense oligodeoxynucleotidetargeted to c-fos mRNA.

Chen et al. (Cancer Lett., 1994, 85, 119) describe repression of c-junexpression by antisense oligodeoxynucleotides resulting in theinhibition of cell proliferation in E5a transformed cells.

Gillardon et al. describe the topical application of c-fos antisenseoligodeoxynucleotides to the rat spinal cord (Eur. J. Neurosci., 1994,6, 880) ultraviolet (UV)-exposed rat eyes (British J. Ophthal., 1995,79, 277) and UV-irradiated rat skin (Carcinogenesis, 1995, 16, 1853).

U.S. Pat. No. 5,602,156, which issued Feb. 11, 1997, to Kohn et al.,discloses non-oligonucleotide compositions and methods for inhibitingthe expression of two metalloproteinases, MMP-1 and MMP-2.

International Publication Number WO 95/02051, published Jan. 19, 1995,discloses antisense oligonucleotides targeted to the mRNA of c-fos andc-jun.

International Publication Number WO 95/03323, published Feb. 2, 1995,discloses antisense nucleic acids which are complementary to thepolynucleotide encoding a polypeptide which is capable ofphosphorylating the c-jun N-terminal activation domain. Also providedare methods for treating a cell proliferative disorder associated withsaid polypeptide.

International Publication Number WO 95/03324, published Feb. 2, 1995,describes a polypeptide which phosphorylates the c-jun N-terminalactivation domain. This publication also discloses a polynucleotidesequence encoding the polypeptide.

To date, there are no known therapeutic agents which effectively inhibitgene expression of c-fos and/or c-jun. Furthermore, there are to date noknown therapeutic agents that modulate the metastasis of malignantcells. The compositions and methods of the invention overcome theselimitations. Further objectives of the invention are apparent from thepresent disclosure.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are providedwhich specifically hybridize with nucleic acids encoding c-Fos or c-Jun.Certain oligonucleotides of the invention are designed to bind eitherdirectly to MRNA transcribed from, or to a selected DNA portion of, therespective gene, thereby modulating the amount of protein translatedfrom a c-fos or c-jun mRNA and/or the amount of mRNA transcribed from ac-fos or c-jun gene, respectively. Such modulation can, in turn, effectthe modulation of enzymes and cellular processes involved in themetastasis of malignant cells.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents.

It has been discovered that the c-fos and c-jun genes, encoding thec-Fos and c-Jun proteins, respectively, are particularly amenable tothis approach. As a consequence of the association between cellularproliferation and AP-1 (the heterodimer of c-Fos and c-Jun) expression,modulation of the expression of c-fos and/or c-jun leads to modulationof AP-1, and, accordingly, modulation of cellular proliferation. Suchmodulation is desirable for treating or modulating varioushyperproliferative disorders or diseases, such as various cancers. Suchinhibition is further desirable for preventing or modulating thedevelopment of such diseases or disorders in an animal suspected ofbeing, or known to be, prone to such diseases or disorders. If desired,modulation of one subunit can be combined with modulation of the subunitof AP-1 in order to achieve a requisite degree of effect uponAP-1-mediated transcription.

Methods of modulating the expression of c-Fos or c-Jun proteinscomprising contacting animals with oligonucleotides specificallyhybridizable with a c-fos or c-jun gene, respectively, are hereinprovided. These methods are believed to be useful both therapeuticallyand diagnostically as a consequence of the association between AP-1expression and cellular proliferation. These methods are also useful astools, for example, in the detection and determination of the role ofAP-1 protein expression in various cell functions and physiologicalprocesses and conditions, and for the diagnosis of conditions associatedwith such expression and activation.

The present invention also comprises methods of inhibiting AP-1-mediatedtranscriptional activation using the oligonucleotides of the invention.Methods of treating conditions in which abnormal or excessiveAP-1-mediated transcriptional activation and cellular proliferationoccur are also provided. These methods employ the oligonucleotides ofthe invention and are believed to be useful both therapeutically and asclinical research and diagnostic tools. The oligonucleotides of thepresent invention may also be used for research purposes. Thus, thespecific hybridization exhibited by the oligonucleotides of the resentinvention may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

Methods comprising contacting animals with oligonucleotides specificallyhybridizable with nucleic acids encoding c-Fos or c-Jun proteins areherein provided. Such methods can be used to modulate or detect theexpression of c-fos or c-jun genes and are thus believed to be usefulboth therapeutically and diagnostically.

The methods disclosed herein are also useful, for example, as clinicalresearch tools in the detection and determination of the role ofAP-1-mediated gene expression in various immune system functions andphysiological processes and conditions, and for the diagnosis ofconditions associated with their expression. The specific hybridizationexhibited by the oligonucleotides of the present invention may be usedfor assays, purifications, cellular product preparations and in othermethodologies which may be appreciated by persons of ordinary skill inthe art. For example, because the oligonucleotides of this inventionspecifically hybridize to nucleic acids encoding c-Fos or c-Jun,sandwich and other assays can easily be constructed to exploit thisfact. Detection of specific hybridization of an oligonucleotide of theinvention with a nucleic acid encoding a c-Fos or c-Jun protein presentin a sample can routinely be accomplished Such detection may includedetectably labeling an oligonucleotide of the invention by enzymeconjugation, radiolabeling or any other suitable detection system. Anumber of assays may be formulated employing the present invention,which assays will commonly comprise contacting a tissue or cell samplewith a detectably labeled oligonucleotide of the present invention underconditions selected to permit hybridization and measuring suchhybridization by detection of the label, as is appreciated by those ofordinary skill in the art.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligonucleotides for use in antisenseinhibition of the function of RNA and DNA encoding a c-Fos protein or ac-Jun protein. The present invention also employs oligonucleotides whichare designed to be specifically hybridizable to DNA or messenger RNA(mRNA) encoding such proteins and ultimately modulating the amount ofsuch proteins transcribed from their respective genes. Suchhybridization with mRNA interferes with the normal role of mRNA andcauses a modulation of its function in cells. The functions of mRNA tobe interfered with include all vital functions such as translocation ofthe RNA to the site for protein translation, actual translation ofprotein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and possibly even independent catalytic activity which may beengaged in by the RNA. The overall effect of such interference with mRNAfunction is modulation of the expression of a c-Fos protein or a c-Junprotein. In the context of this invention, “modulation” means either anincrease (stimulation) or a decrease (inhibition) in the expression of agene. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.This term includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas oligonucleotides having non-naturally-occurring portions whichfunction similarly. Such modified or substituted oligonucleotides areoften preferred over native forms because of desirable properties suchas, for example, enhanced cellular uptake, enhanced binding to targetand increased stability in the presence of nucleases.

An oligonucleotide is a polymer of a repeating unit generically known asa nucleotide. An unmodified (naturally occurring) nucleotide has threecomponents: (1) a nitrogenous base linked by one of its nitrogen atomsto (2) a 5-carbon cyclic sugar and (3) a phosphate, esterified to carbon5 of the sugar. When incorporated into an oligonucleotide chain, thephosphate of a first nucleotide is also esterified to carbon 3 of thesugar of a second, adjacent nucleotide. The “backbone” of an unmodifiedoligonucleotide consists of (2) and (3), that is, sugars linked togetherby phosphodiester linkages between the carbon 5 (5′) position of thesugar of a first nucleotide and the carbon 3 (3′) position of a second,adjacent nucleotide. A “nucleoside” is the combination of (1) anucleobase and (2) a sugar in the absence of (3) a phosphate moiety(Kornberg, A.,DNA Replication, pp. 4-7, W. H. Freeman & Co., SanFrancisco, 1980). The backbone of an oligonucleotide positions a seriesof bases in a specific order; the written representation of this seriesof bases, which is conventionally written in 5′ to 3′ order, is known asa nucleotide sequence. The oligonucleotides in accordance with thisinvention preferably comprise from about 8 to about 30 nucleotides. Itis more preferred that such oligonucleotides comprise from about 15 to25 nucleotides.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” Antisense oligonucleotides are commonly used as researchreagents, diagnostic aids, and therapeutic agents. For example,antisense oligonucleotides, which are able to inhibit gene expressionwith exquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses.

The specificity and sensitivity of oligonucleotides is also harnessed bythose of skill in the art for therapeutic uses. For example, thefollowing U.S. patents demonstrate palliative, therapeutic and othermethods utilizing antisense oligonucleotides. U.S. Pat. No. 5,135,917provides antisense oligonucleotides that inhibit human interleukin-1receptor expression. U.S. Pat. No. 5,098,890 is directed to antisenseoligonucleotides complementary to the c-myb oncogene and antisenseoligonucleotide therapies for certain cancerous conditions. U.S. Pat.No. 5,087,617 provides methods for treating cancer patients withantisense oligonucleotides. U.S. Pat. No. 5,166,195 providesoligonucleotide inhibitors of Human Immunodeficiency Virus (HIV). U.S.Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No.5,194,428 provides antisense oligonucleotides having antiviral activityagainst influenzavirus. U.S. Pat. No. 4,806,463 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementarybase sequence to a portion of an oncogene. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotideanalogs used to prevent replication of foreign nucleic acids in cells.U.S. Pat. No. 4,689,320 is directed to antisense oligonucleotides asantiviral agents specific to cytomegalovirus (CMV). U.S. Pat.No.5,098,890 provides oligonucleotides complementary to at least aportion of the mRNA transcript of the human c-myb gene. U.S. Pat. No.5,242,906 provides antisense oligonucleotides useful in the treatment oflatent Epstein-Barr virus (EBV) infections.

It is preferred to target specific genes for antisense attack.“Targeting” an oligonucleotide to the associated nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene (c-fos or c-jun) encoding a subunit of AP-1, for which modulationis desired in certain instances. The targeting process also includesdetermination of a region (or regions) within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult. Once the target region have been identified, oligonucleotidesare chosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity to give thedesired effect.

There are many regions of a gene that may be targeted for antisensemodulation: the region of the 5′ Cap, a specialized structure that atleast partially mediates ribosome binding; the 5′ untranslated(noncoding) region (hereinafter, the “5′-UTR”); the translationinitiation codon region (hereinafter, the “tIR”); the open reading frame(hereinafter, the “ORF”); the translation termination codon region(hereinafter, the “tTR”); and the 31′ untranslated (noncoding) region(hereinafter, the “3′-UTR”), which has at its 3′ end a “poly A” tail. Asis known in the art, these regions are arranged in a typical messengerRNA molecule in the following order (left to right, 5′ to 3′): 5′ Cap,5′-UTR, tIR, ORF, tTR, 3′-UTR, poly A tail. As is also known in the art,although some eukaryotic transcripts are directly translated, many ORFscontain one or more sequences, known as “introns,” which are excisedfrom a transcript before it is translated; the expressed (unexcised)portions of the ORF are referred to as “exons” (Alberts et al.,Molecular Biology of the Cell, pp. 331-332 and 411-415, GarlandPublishing Inc., New York, 1983). Furthermore, because many eukaryoticORFs are a thousand nucleotides or more in length, it is oftenconvenient to subdivide the ORF into, e.g., the 5′ ORF region, thecentral ORF region, and the 3′ ORF region. In some instances, an ORFcontains one or more sites that may be targeted due to some functionalsignificance in vivo. Examples of the latter types of sites includeintragenic stem-loop structures (see, e.g., U.S. Pat. No. 5,512,438)and, in unprocessed mRNA molecules, intron/exon splice sites. Within thecontext of the present invention, one preferred intragenic site is theregion encompassing the translation initiation codon of the open readingframe (ORF) of the gene. Because, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon.” A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo.Furthermore, 5′-UUU functions as a translation initiation codon in vitro(Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al., Somat.Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, Chapter 78 in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, Vol. 2, p. 1303, Neidhardt et al., eds., American Society forMicrobiology, Washington, D.C., 1987). Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (prokaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes may have two ormore alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions, in order to generate relatedpolypeptides having different amino terminal sequences (Markussen etal., Development, 1995, 121, 3723; Gao et al., Cancer Res., 1995, 55,743; McDermott et al., Gene, 1992, 117, 193; Perri et al., J. Biol.Chem., 1991, 266, 12536; French et al., J. Virol., 1989, 63, 3270;Pushpa-Rekha et al., J. Biol. Chem., 1995, 270, 26993; Monaco et al., J.Biol. Chem., 1994, 269, 347; DeVirgilio et al., Yeast, 1992, 8, 1043;Kanagasundaram et al., Biochim. Biophys. Acta, 1992, 1171, 198; Olsen etal., Mol. Endocrinol., 1991, 5, 1246; Saul et al., Appl. Environ.Microbiol., 1990, 56, 3117; Yaoita et al., Proc. Natl. Acad. Sci. USA,1990, 87, 7090; Rogers et al., EMBO J., 1990, 9, 2273). In the contextof the invention, “start codon” and “translation initiation codon” referto the codon or codons that are used in vivo to initiate translation ofan mRNA molecule transcribed from a gene encoding a c-Fos or c-Junprotein, regardless of the sequence(s) of such codons. It is also knownin the art that a translation termination codon (or “stop codon”) of agene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA(the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The terms “start codon region” and “translationinitiation region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation termination codon.

In the context of this invention, the term “oligonucleotide” includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent intersugar (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides may be preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced binding to target and increased stability inthe presence of nucleases.

Specific examples of some preferred modified oligonucleotides envisionedfor this invention include those containing phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioates and those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ [knownas a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH₂). Also preferred areoligonucleotides having morpholino backbone structures (Summerton andWeller, U.S. Pat. No. 5,034,506). Further preferred are oligonucleotideswith NR—C(*)—CH₂—CH₂, CH₂—NR—C(*)—CH₂, CH₂—CH₂—NR—C(*), C(*)—NR—CH₂—CH₂and CH₂—C(*)—NR—CH₂ backbones, wherein “*” represents O or S (known asamide backbones; DeMesmaeker et al., WO 92/20823, published Nov. 26,1992). In other preferred embodiments, such as the peptide nucleic acid(PNA) backbone, the phosphodiester backbone of the oligonucleotide isreplaced with a polyamide backbone, the nucleobases being bound directlyor indirectly to the aza nitrogen atoms of the polyamide backbone(Nielsen et al., Science, 1991, 254, 1497; U.S. Pat. No. 5,539,082).

The oligonucleotides of the invention may additionally or alternativelyinclude nucleobase modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include adenine (A), guanine (G),thymine (T), cytosine (C) and uracil (U). Modified nucleobases includenucleobases found only infrequently or transiently in natural nucleicacids, e.g., hypoxanthine, 6-methyladenine, 5-methylcytosine,5-hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, aswell synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N⁶(6-aminohexyl)adenine and 2,6-diaminopurine (Kornberg,A., DNA Replication, pp. 75-77, W. H. Freeman & Co., San Francisco,1980; Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513).

The oligonucleotides of the invention may additionally or alternativelycomprise substitutions of the sugar portion of the individualnucleotides. For example, oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group. Otherpreferred modified oligonucleotides may contain one or more substitutedsugar moieties comprising one of the following at the 2′ position: OH,SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ orO(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN;CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a reporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A preferred modification,particularly for orally deliverable pharmaceutical compositions, is2′-methoxyethoxy [2′—O—CH₂CH₂OCH₃, also known as 2′—O—(2-methoxyethyl)](Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferredmodifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2-OCH₂CH₂CH₃)and 2′-fluoro (2′-F).

Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. The5′ and 3′ termini of an oligonucleotide may also be modified to serve aspoints of chemical conjugation of, e.g., lipophilic moieties (seeimmediately subsequent paragraph), intercalating agents (Kuyavin et al.,WO 96/32496, published Oct. 17, 1996; Nguyen et al., U.S. Pat. No.4,835,263, issued May 30, 1989) or hydroxyalkyl groups (Helene et al.,WO 96/34008, published Oct. 31, 1996).

Other positions within an oligonucleotide of the invention can be usedto chemically link thereto one or more effector groups to form anoligonucleotide conjugate. An “effector group” is a chemical moiety thatis capable of carrying out a particular chemical or biological function.Examples of such effector groups include, but are not limited to, an RNAcleaving group, a reporter group, an intercalator, a group for improvingthe pharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A variety of chemical linkersmay be used to conjugate an effector group to an oligonucleotide of theinvention. As an example, U.S. Pat. No. 5,578,718 to Cook et al.discloses methods of attaching an alkylthio linker, which may be furtherderivatized to include additional groups, to ribofuranosyl positions,nucleosidic base positions, or on internucleoside linkages. Additionalmethods of conjugating oligonucleotides to various effector groups areknown in the art; see, e.g., Protocols for Oligonucleotide Conjugates(Methods in Molecular Biology, Volume 26) Agrawal, S., ed., HumanaPress, Totowa, N.J., 1994.

Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention(Gebeyehu et al., Nucleic Acids Res., 1987, 15, 4513). Such lipophilicmoieties include but are not limited to a cholesteryl moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides, are disclosed in U.S. Pat. Nos. 5,138,045, 5,218,105and 5,459,255, the contents of which are hereby incorporated byreference.

The oligonucleotides of the invention may additionally or alternativelybe prepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides ofthe invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate]derivatives according to the methods disclosed in WO 93/24510 toGosselin et al., published Dec. 9, 1993.

The present invention also includes oligonucleotides that aresubstantially chirally pure with regard to particular positions withinthe oligonucleotides. Examples of substantially chirally pureoligonucleotides include, but are not limited to, those havingphosphorothioate linkages that are at least 75% Sp or Rp (Cook et al.,U.S. Pat. No. 5,587,361, issued Dec. 24, 1996) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoamidateor phosphotriester linkages (Cook, U.S. Pat. No. 5,212,295, issued May18, 1993; Cook, U.S. Pat. No. 5,521,302, issued May 28, 1996).

The present invention also includes oligonucleotides which are chimericoligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. Byway of example, such “chimeras” may be “gapmers,” i.e., oligonucleotidesin which a central portion (the “gap”) of the oligonucleotide serves asa substrate for, e.g., RNase H, and the 5′ and 3′ portions (the “wings”)are modified in such a fashion so as to have greater affinity for thetarget RNA molecule but are unable to support nuclease activity (e.g.,2′-fluoro- or 2′-methoxyethoxy substituted). Other chimeras include“wingmers,” that is, oligonucleotides in which the 5′ portion of theoligonucleotide serves as a substrate for, e.g., RNase H, whereas the 3′portion is modified in such a fashion so as to have greater affinity forthe target RNA molecule but is unable to support nuclease activity(e.g., 2′-fluoro- or 2′-methoxyethoxy substituted), or vice-versa.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. Teachings regarding thesynthesis of particular modified oligonucleotides are herebyincorporated by reference from the following U.S. patents or pendingpatent applications, each of which is commonly assigned with thisapplication: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingb-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties, attached at any of avariety of positions of the nucleoside; and U.S. Pat. No. 5,587,361,drawn to oligonucleotides having phosphorothioate linkages of highchiral purity.

The oligonucleotides of the present invention can be utilized astherapeutic compounds, diagnostic tools and as research reagents andkits. The term “therapeutic uses” is intended to encompass prophylactic,palliative and curative uses wherein the oligonucleotides of theinvention are contacted with animal cells either in vivo or ex vivo.When contacted with animal cells ex vivo, a therapeutic use includesincorporating such cells into an animal after treatment with one or moreoligonucleotides of the invention.

For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a c-Fos or c-Jun protein is, for example, treated byadministering oligonucleotides in accordance with this invention. Theoligonucleotides of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an oligonucleotide to asuitable pharmaceutically acceptable diluent or carrier. Workers in thefield have identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

The oligonucleotides of the present invention can be further used todetect the presence of c-fos- or c-jun-specific nucleic acids in a cellor tissue sample. For example, radiolabeled oligonucleotides can beprepared by ³²P labeling at the 5′ end with polynucleotide kinase(Sambrook et al., Molecular Cloning. A Laboratory Manual, Vol. 2, p.10.59, Cold Spring Harbor Laboratory Press, 1989). Radiolabeledoligonucleotides are then contacted with cell or tissue samplessuspected of containing c-fos or c-jun message RNAs (and thus c-Fos orc-Jun proteins), and the samples are washed to remove unboundoligonucleotide. Radioactivity remaining in the sample indicates thepresence of bound oligonucleotide, which in turn indicates the presenceof nucleic acids complementary to the oligonucleotide, and can bequantitated using a scintillation counter or other routine means.Expression of nucleic acids encoding these proteins is thus detected.

Radiolabeled oligonucleotides of the present invention can also be usedto perform autoradiography of tissues to determine the localization,distribution and quantitation of c-Fos or c-Jun proteins for research,diagnostic or therapeutic purposes. In such studies, tissue sections aretreated with radiolabeled oligonucleotide and washed as described above,then exposed to photographic emulsion according to routineautoradiography procedures. The emulsion, when developed, yields animage of silver grains over the regions expressing a c-Fos or c-Jungene. Quantitation of the silver grains permits detection of theexpression of mRNA molecules encoding these proteins and permitstargeting of oligonucleotides to these areas.

Analogous assays for fluorescent detection of expression of c-fos orc-jun nucleic acids can be developed using oligonucleotides of thepresent invention which are conjugated with fluorescein or otherfluorescent tags instead of radiolabeling. Such conjugations areroutinely accomplished during solid phase synthesis usingfluorescently-labeled amidites or controlled pore glass (CPG) columns.Fluorescein-labeled amidites and CPG are available from, e.g., GlenResearch (Sterling, Va.).

The present invention employs oligonucleotides targeted to nucleic acidsencoding c-Fos or c-Jun proteins and oligonucleotides targeted tonucleic acids encoding such proteins. Kits for detecting the presence orabsence of expression of a c-Fos and/or c-Jun protein may also beprepared. Such kits include an oligonucleotide targeted to anappropriate gene, i.e., a gene encoding a c-Fos or c-Jun protein.Appropriate kit and assay formats, such as, e.g., “sandwich” assays, areknown in the art and can easily be adapted for use with theoligonucleotides of the invention. Hybridization of the oligonucleotidesof the invention with a nucleic acid encoding a c-Fos or c-Jun proteincan be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection systems. Kits fordetecting the presence or absence of a c-Fos or c-Jun protein may alsobe prepared.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleotides. For example,adenine and thymine are complementary nucleobases which pair through theformation of hydrogen bonds. “Complementary,” as used herein, refers tothe capacity for precise pairing between two nucleotides. For example,if a nucleotide at a certain position of an oligonucleotide is capableof hydrogen bonding with a nucleotide at the same position of a DNA orRNA molecule, then the oligonucleotide and the DNA or RNA are consideredto be complementary to each other at that position. The oligonucleotideand the DNA or RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides which can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity or precise pairing such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. It is understood in the art that an oligonucleotide need notbe 100% complementary to its target DNA sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target DNA or RNA moleculeinterferes with the normal function of the target DNA or RNA to cause adecrease or loss of function, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered an oligonucleotide in accordance with the invention,commonly in a pharmaceutically acceptable carrier, in doses ranging from0.01 ug to 100 g per kg of body weight depending on the age of thepatient and the severity of the disorder or disease state being treated.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of theoligonucleotide may either be increased in the event the patient doesnot respond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disorder or diseasestate is observed, or if the disorder or disease state has been abated.

In some cases it may be more effective to treat a patient with anoligonucleotide of the invention in conjunction with other traditionaltherapeutic modalities in order to increase the efficacy of a treatmentregimen. In the context of the invention, the term “treatment regimen”is meant to encompass therapeutic, palliative and prophylacticmodalities. For example, a patient may be treated with conventionalchemotherapeutic agents, particularly those used for tumor and cancertreatment. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds.,Rahay, N.J., 1987). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

In another preferred embodiment of the invention, a first antisenseoligonucleotide targeted to c-fos is used -in combination with a secondantisense oligonucleotide targeted to c-jun in order to modulate AP-1molecules to a more extensive degree than can be achieved when eitheroligonucleotide used individually.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 ug to 100 g per kg of body weight, once or more daily,to once every 20 years. In the case of in individual known or suspectedof being prone to a neoplastic or malignant condition, prophylacticeffects may be achieved by administration of preventative doses, rangingfrom 0.01 ug to 100 g per kg of body weight, once or more daily, to onceevery 20 years.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated. Withinthe context of the invention, “administration” indicates the topical(including ophthalmic, vaginal, rectal, intranasal, transdermal), oralor parenteral contacting of an oligonucleotide, or pharmaceuticalcomposition comprising an oligonucleotide, with cells, tissues or organsof a mammal including a human. Parenteral administration includesintravenous drip; subcutaneous, intraperitoneal, intravitreal orintramuscular injection; and intrathecal or intraventricularadministration as herein described.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, nucleic acidcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable in certain instances. Topical administration alsoincludes the delivery of oligonucleotides into the epidermis of a mammalby electroporation (Zewert et al., WO 96/39531, published Dec. 12,1996).

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Intravitreal injection, for the direct delivery of drug to the vitreoushumor of a mammalian eye, is described in U.S. Pat. No. 5,595,978, whichissued Jan. 21, 199, and which is assigned to the same assignee as theinstant application, the contents of which are hereby incorporated byreference.

Intraluminal drug administration, for the direct delivery of drug to anisolated portion of a tubular organ or tissue (e.g., such as an artery,vein, ureter or urethra), may be desired for the treatment of patientswith diseases or conditions afflicting the lumen of such organs ortissues. To effect this mode of oligonucleotide administration, acatheter or cannula is surgically introduced by appropriate means. Forexample, for treatment of the left common carotid artery, a cannula isinserted thereinto via the external carotid artery. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising the oligonucleotides of the invention is infusedthrough the cannula or catheter into the isolated segment. Afterincubation for from about 1 to about 120 minutes, during which theoligonucleotide is taken up by cells of the interior lumen of thevessel, the infusion cannula or catheter is removed and flow within thetubular organ or tissue is restored by removal of the ligatures whicheffected the isolation of a segment thereof (Morishita et al., Proc.Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474). Antisense oligonucleotidesmay also be combined with a biocompatible matrix or carrier, such as ahydrogel material, and applied directly to vascular tissue in vivo(Rosenberg et al ., U.S. Pat. No. 5,593,974, issued Jan. 14, 1997).

Intraventricular drug administration, for the direct delivery of drug tothe brain of a patient, may be desired for the treatment of patientswith diseases or conditions afflicting the brain. To effect this mode ofoligonucleotide administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region (Zimmet al., Cancer Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72(11Suppl.), 3416). The pump is used to inject the oligonucleotides andallows precise dosage adjustments and variation in dosage schedules withthe aid of an external programming device. The reservoir capacity of thepump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1 mL/h.Depending on the frequency of administration, ranging from daily tomonthly, and the dose of drug to be administered, ranging from 0.01 μgto 100 g per kg of body weight, the pump reservoir may be refilled at3-10 week intervals. Refilling of the pump is accomplished bypercutaneous puncture of the self-sealing septum of the pump.

Intrathecal drug administration for the introduction of drug into thespinal column of a patient may be desired for the treatment of patientswith diseases of the central nervous system. To effect this route ofoligonucleotide administration, a silicon catheter is surgicallyimplanted into the L3-4 lumbar spinal interspace of a human patient, andis connected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region (Luer and Hatton, The Annals ofPharmacotherapy, 1993, 27, 912; Ettinger et al., 1978, Cancer, 41, 1270,1978; Yaida et al. , Regul. Pept., 1995, 59, 193). The pump is used toinject the oligonucleotides and allows precise dosage adjustments andvariations in dose schedules with the aid of an external programmingdevice. The reservoir capacity of the pump is 18-20 mL, and infusionrates may vary from 0.1 mL/h to 1 mL/h. Depending on the frequency ofdrug administration, ranging from daily to monthly, and dosage of drugto be administered, ranging from 0.01 μg to 100 g per kg of body weight,the pump reservoir may be refilled at 3-10 week intervals. Refilling ofthe pump is accomplished by a single percutaneous puncture to theself-sealing septum of the pump. The distribution, stability andpharmacokinetics of oligonucleotides within the central nervous systemmay be followed according to known methods (Whitesell et al., Proc.Natl. Acad. Sci. (USA), 1993, 90, 4665).

To effect delivery of oligonucleotides to areas other than the brain orspinal column via this method, the silicon catheter is configured toconnect the subcutaneous infusion pump to, e-g., the hepatic artery, fordelivery to the liver (Kemeny et al., Cancer, 1993, 71:1964). Infusionpumps may also be used to effect systemic delivery of oligonucleotides(Ewel et al., Cancer Research, 1992, 52:3005; Rubenstein et al., J.Surg. Oncol., 1996, 62:194).

Regardless of the method by which the oligonucleotides of the inventionare introduced into a patient, colloidal dispersion systems may be usedas delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles and liposomes. A preferred colloidal dispersionsystem is a plurality of liposomes, artificial membrane vesicles whichmay be used as cellular delivery vehicles for bioactive agents in vitroand in vivo (Mannino et al., Biotechniques, 1988, 6, 682; Blume andCevc, Biochem. et Biophys. Acta, 1990, 1029, 91; Lappalainen et al.,Antiviral Res., 1994, 23, 119; Chonn and Cullis, Current Op. Biotech.,1995, 6, 698). It has been shown that large unilamellar vesicles (LUV),which range in size from 0.2-0.4 μm, can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand delivered to brain cells in a biologically active form (Fraley etal., Trends Biochein. Sci., 1981, 6, 77). The composition of theliposome is usually a combination of lipids, particularly phospholipids,in particular, high phase transition temperature phospholipids, usuallyin combination with one or more steroids, particularly cholesterol.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipidis,cerebrosides and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbonatoms, particularly from 16-18 carbon atoms, and is saturated (lackingdouble bonds within the 14-18 carbon atom chain). Illustrativephospholipids include phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of colloidal dispersion systems, including liposomes, canbe either passive or active. Passive targeting utilizes the naturaltendency of liposomes to distribute to cells of the reticuloendothelialsystem in organs that contain sinusoidal capillaries. Active targeting,by contrast, involves modification of the liposome by coupling thereto aspecific ligand such as a viral protein coat (Morishita et al., Proc.Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474), monoclonal antibody (or asuitable binding portion thereof), sugar, glycolipid or protein (or asuitable oligopeptide fragment thereof), or by changing the compositionand/or size of the liposome in order to achieve distribution to organsand cell types other than the naturally occurring sites of localization.The surface of the targeted colloidal dispersion system can be modifiedin a variety of ways. In the case of a liposomal targeted deliverysystem, lipid groups can be incorporated into the lipid bilayer of theliposome in order to maintain the targeting ligand in close associationwith the lipid bilayer. Various linking groups can be used for joiningthe lipid chains to the targeting ligand. The targeting ligand, whichbinds a specific cell surface molecule found predominantly on cells towhich delivery of the oligonucleotides of the invention is desired, maybe, for example, (1) a hormone, growth factor or a suitable oligopeptidefragment thereof which is bound by a specific cellular receptorpredominantly expressed by cells to which delivery is desired or (2) apolyclonal or monoclonal antibody, or a suitable fragment thereof (e.g.,Fab; F(ab′)₂) which specifically binds an antigenic epitope foundpredominantly on targeted cells. Two or more bioactive agents (e.g., anoligonucleotide and a conventional drug; two oligonucleotides) can becombined within, and delivered by, a single liposome. It is alsopossible to add agents to colloidal dispersion systems which enhance theintercellular stability and/or targeting of the contents thereof.

Compositions for parehteral, intrathecal or intraventricularadministration, or colloidal dispersion systems, may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. Dosing is dependent on severity and responsivenessof the disease state to be treated, with the course of treatment lastingfrom several days to several months, or until a cures is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years.

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

EXAMPLES Example 1 Chemical Synthesis and Nucleotide Sequence ofOligonucleotides

General Synthetic Techniques:

Oligonucleotides were synthesized on an automated DNA synthesizer usingstandard phosphoramidite chemistry with oxidation using iodine.β-Cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages.

The synthesis of 2′-O-methyl- (a.k.a. 2′-methoxy-) phosphorothioateoligonucleotides was according to the procedures set forth abovesubstituting 2′-O-methyl β-cyanoethyldiisopropyl phosphoramidites(Chemgenes, Needham, Mass.) for standard phosphoramidites and increasingthe wait cycle after the pulse delivery of tetrazole and base to 360seconds.

Similarly, 2′-O-propyl- (a.k.a 2′-propoxy-) phosphorothioateoligonucleotides were prepared by slight modifications of this procedureand essentially according to procedures disclosed in U.S. patentapplication Ser. No. 08/383,666, filed Feb. 3, 1995, which is assignedto the same assignee as the instant application and which isincorporated by reference herein.

The 2′-fluoro-phosphorothioate oligonucleotides of the invention weresynthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared asdisclosed in U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both ofwhich are assigned to the same assignee as the instant application andwhich are incorporated by reference herein. The2′-fluoro-oligonucleotides were prepared using phosphoramidite chemistryand a slight modification of the standard DNA synthesis protocol (i.e.,deprotection was effected using methanolic ammonia at room temperature).

The 2′-methoxyethoxy oligonucleotides were synthesized essentiallyaccording to the methods of Martin et al. (Helv. Chim. Acta, 1995, 78,486). For ease of synthesis, the 3′ nucleotide of the 2′-methoxyethoxyoligonucleotides was a deoxynucleotide, and 2′-O—CH₂CH₂OCH³⁻cytosineswere 5-methyl cytosines, which were synthesized according to theprocedures described below.

PNA antisense analogs were prepared essentially as described in U.S.Pat. Nos. 5,539,082 and 5,539,083, both of which (1) issued Jul. 23,1996, (2) are assigned to the same assignee as the instant applicationand (3) are incorporated by reference herein.

Oligonucleotides comprising 2,6-diaminopurine were prepared essentiallyas described in U.S. Pat. No. 5,506,351 which issued Apr. 9, 1996, isassigned to the same assignee as the instant application and which isincorporated by reference herein. Oligonucleotides comprising2,6-diaminopurine can also be prepared by enzymatic means (Bailly etal., Proc. Natl. Acad. Sci. U.S.A., 1996, 93:13623).

After cleavage from the controlled pore glass (CPG) column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide, at 55° C.for 18 hours, the oligonucleotides were purified by precipitation 2×from 0.5 M NaCl with 2.5 volumes of ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea and 45 mMTris-borate buffer (pH 7).

Synthesis of 5-Methyl Cytosine Monomers

2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine:

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2′-O-Methoxyethyl-5′-O-dinethoxytrityl-5-methyluridine:

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

2′-0-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approximately90% product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxy-trityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. Methanol (400 mL) saturated with NH3 gas was added and thevessel heated to 100° C. for 2 hours (thin layer chromatography, tic,showed complete conversion). The vessel contents were evaporated todryness and the residue was dissolved in EtOAc (500 mL) and washed oncewith saturated NaCl (200 mL). The organics were dried over sodiumsulfate and the solvent was evaporated to give 85 g (95%) of the titlecompound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tic showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N⁴-Benzoyl-2-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tic showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

Nucleotide Sequences:

Table 1 shows the sequence and activity of each of the oligonucleotidesevaluated for inhibition of c-jun mRNA expression, and Table 2 shows thesequence and activity of the oligonucleotides evaluated for inhibitionof c-fos mRNA expression. Oligonucleotide activities were evaluated asdescribed infra in Example 2 et. seq. For the nucleotide sequence of thehuman c-jun gene, see Hattori et al., Proc. Natl. Acad. Sci. U.S.A.,1988, 85:9148, and Genbank accession No. J04111 (“HUMJUNA”). Thenucleotide sequence of the human c-fos gene is described by Van Straatenet al., Proc. Natl. Acad. Sci. U.S.A., 1983, 80:3183, and in Genbankaccession No. K00650 (“HUMFOS”).

TABLE 1 Phosphorothioate Oligonucleotides Targeted to Human c-jun TARGET% ISIS # SEQUENCE SEQ ID NO: REGION CONTROL* 10570GCC-ACA-CTC-AGT-GCA-ACT-CT 1 5′ Cap 62 10571 CGC-ACC-TCC-ACT-CCC-GCC-TC2 5′-UTR 100 10572 ACC-AGC-CCG-GGA-GCC-ACA-GG 3 5′-UTR 39 10578GCT-GCG-CCG-CCG-ACG-TGA-CG 4 ORF 37 10579 CGC-CCC-GCC-GCC-GCT-GCT-CA 5ORF 41 10580 GTG-TCT-CGC-CGG-GCA-TCT-CG 6 ORF 19 10581CCC-CCG-ACG-GTC-TCT-CTT-CA 7 tTR 24 10582 TCA-GCC-CCC-GAC-GGT-CTC-TC 83′-UTR 17 10583 TGC-CCC-TCA-GCC-CCC-GAC-GG 9 3′-UTR 20 13305TGC-GGG-TGA-GTG-GTA-G 118 ORF N.D.** 10582 Controls: 10582TCA-GCC-CCC-GAC-GGT-CTC-TC 8 3′-UTR 11562 GAG-AGA-CCG-TCG-GGG-GCT-GA 29sense control 11563 CAC-CTC-CAC-GCG-CTT-CTG-GC 30 scrambled control11564 TCG-GCA-CCT-GAA-GGA-CTT-TC 31 mismatch control *Control is TPAinduction, at 1 hour, in A549 cells. **N.D., not determined.

TABLE 2 Phosphorothioate Oligonucleotides Targeted to Human c-fos TARGETISIS # SEQUENCE SEQ ID NO: REGION % CONTROL* 10628TGC-TCG-CTG-CAG-ATG-CGG-TT 10 5′Cap 79 10629 CGG-TCA-CTG-CTC-GTT-CGC-TG11 5′-UTR 72 10630 CAT-CGT-GGC-GGT-TAG-GCA-AA 12 tIR 91 10631GAG-AAC-ATC-ATC-GTG-GCG-GT 13 tIR 118 10632 ACC-GTG-GGA-ATG-AAG-TTG-GC14 ORF 63 10633 AGC-TCC-CTC-CTC-CGG-TTG-CG 15 ORF 24 10634TTG-CAG-GCA-GGT-CGG-TGA-GC 16 ORF 42 10635 TGG-CAC-GGA-GCG-GGC-TGT-CT 17ORF 12 10636 TGC-TGC-TGC-CCT-TGC-GGT-GG 18 ORF 42 10637CCT-CAC-AGG-GCC-AGC-AGC-GT 19 tTR 35 10638 GGT-GCC-GGC-TGC-CTC-CCC-TT 203′-UTR 22 10639 AAG-TCC-TTG-AGG-CCC-ACA-GC 21 3′-UTR 9 10640CCC-CTC-CAG-CAG-CTA-CCC-TT 22 3′-UTR 87 10641 TCC-CGT-CCC-CAG-AAG-CAG-TA23 3′-UTR 68 10642 CGC-GCC-CGG-CCT-GAA-AAT-TT 24 3′-UTR 87 10643CCT-GCC-TCG-GCC-TCC-CAA-AG 25 3′-UTR 39 10644 CCC-CCA-CTT-CCG-CCC-ACT-AT26 3′-UTR 104 10645 TGG-TGC-CTG-CGT-GAT-ACT-CG 27 3′-UTR 56 10646CCC-TCC-CAG-GCT-CAA-GTC-AT 28 3′-UTR 100 10639 Controls: 10639AAG-TCC-TTG-AGG-CCC-ACA-GC 21 3′-UTR 11184 GCT-GTG-GGC-CTC-AAG-GAC-TT 32sense control 11185 ATG-TGC-TAG-ATG-CGC-AAA-GT 33 mismatch control 11186ACG-TCC-GAT-TCC-GAG-CGC-AA 34 scrambled control 11187CAG-TGG-CCA-TCA-AAC-CCG-TG 35 scrambled control *Control is TPAinduction, at 1 hour, in A549 cells.

Example 2 Screening for Oligonucleotides that Modulate mRNA Expressionof the AP-1 Subunits c-fos and c-jun

In order to evaluate the activity of potential c-fos and c-junmodulating oligonucleotides, A549 cells were grown in T-75 flasks until80-90% confluent. (Cell line A549 is available from, inter alia, theAmerican Type Culture Collection, Rockville, Md., as ATCC No. CCL-185.)At this time, the cells were washed twice with 10 mL of media (DMEM),followed by the addition of 5 mL of DMEM containing 20 μg/mL ofLIPOFECTIN™ (i.e., DOTMA/DOPE(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammoniumchloride/dioleoylphosphatidyl ethanolamine)). The oligonucleotides wereadded from a 10 μM stock solution to a final concentration of 400 nM,and the two solutions were mixed by swirling the flasks. After 4 hoursat 37° C., the medium was replaced with DMEM containing 10% serum. Atthis point, 1 μM 12-O-tetradecanoylphorbol 13-acetate (TPA) was added toinduce expression of c-fos and c-jun. Cells were extracted inguanidinium one hour later, and the c-fos and c-jun mRNA expression wasdetermined by Northern blotting. Probes for human c-jun and c-fos werePCR products prepared using primers based on the published sequencesthereof (respectively, Hattori et al., Proc. Natl. Acad. Sci. U.S.A.,1988, 85:9148, and Van Straaten et al., Proc. Natl. Acad. Sci. U.S.A.,1983, 80:3183).

As described in Table 1, for inhibiting c-jun mRNA expression, ISIS10580, 10581, 10582 and 10583 were most active (81%, 76%, 83% and 80%inhibition, respectively) Treatment of cells with these oligonucleotidesreduced c-jun expression to 19%, 24%, 17% and 20% (81%, 76%, 83% and 80%inhibition), respectively, of the level seen in control experiments(100% expression, 0% inhibition). These oligonucleotides effectsignificant inhibition of c-jun and are therefor preferred. Basal levelsof c-jun mRNA are typically about 30% of the control value; ISIS 10572,10578 and 10579 reduce c-jun levels to near basal levels (39%, 37% and41%, respectively) and are thus also preferred.

As described in Table 2, the oligonucleotides most effective in reducingc-fos mRNA expression are ISIS 10633, 10635, 10638 and 10639. Treatmentof cells with these oligonucleotides reduced c-fos expression to 24%,12%, 22% and 9% (76%, 88%, 78% and 91% inhibition), respectively, of thelevel seen in control experiments (100% expression, 0% inhibition);basal levels of c-fos mRNA are typically about 3% of the control value.These oligonucleotides effect significant inhibition of c-fos and aretherefor preferred.

Example 3 Dose Response and Specificity of Oligonucleotides Targeted toAP-1 Subunits

Dose-response experiments were performed at different oligonucleotideconcentrations to determine the potency (i.e., ability to decreaseexpression of the appropriate mRNA target) of the most active compoundsidentified in the initial screen (Tables 3 and 4). The decreases intarget mRNA expression effected by ISIS 10582 (c-jun) and ISIS 10639(c-fos) are dose-dependent, as shown in Tables 3 and 4, respectively.

TABLE 3 Dose-Response to Oligonucleotides Targeted to c-junOLIGONUCLEOTIDE c-jun mRNA LEVELS TREATMENT CONCENTRATION (% CONTROL)None (basal level) — 31 Control* — 100 TPA + ISIS 10582  50 nM 72 TPA +ISIS 10582 100 nM 45.5 TPA + ISIS 10582 200 nM 29.5 TPA + ISIS 10582 400nM 16 *Control is TPA induction, at 1 hour, in A549 cells.

TABLE 4 Dose-Response to Oligonucleotides Targeted to c-fosOLIGONUCLEOTIDE c-fos mRNA LEVELS TREATMENT CONCENTRATION (% CONTROL)None (basal level) — 3 Control* — 100 TPA + ISIS 10639  50 nM 64 TPA +ISIS 10639 100 nM 46 TPA + ISIS 10639 200 nM 20.5 TPA + ISIS 10639 400nM 9 *Control is TPA induction, at 1 hour, in A549 cells.

The specificity of the oligonucleotide-mediated inhibition of c-fos andc-jun mRNA expression was further examined by determining the effects ofscrambled, 6- or 7-base mismatch and sense control versions of the mostactive oligonucleotides, ISIS 10582 (c-jun) and ISIS 10639 (c-fos). Ascan be seen in Table 5, ISIS 10582 exhibited potent and specificinhibition of c-jun mRNA expression, but ISIS 11562 (sense version ofISIS 10582; SEQ ID NO:29), ISIS 11564 (6 base pair mismatch version ofISIS 10639; SEQ ID NO:31) and ISIS 11563 (scrambled version of ISIS10639; SEQ ID NO:30) had no detectable effect on c-jun mRNA levelsduring TPA induction (the sequences of ISIS 11562-11564 are given inTable 1).

As can further be seen in Table 5, ISIS 10639 exhibited potent andspecific inhibition of c-fos MRNA expression, but ISIS 11184 (senseversion of ISIS 10639; SEQ ID NO:32), ISIS 11185 (7 base pair mismatchversion of ISIS 10639; SEQ ID NO:33) and ISIS 11186 (scrambled versionof ISIS 10639; SEQ ID NO:34) had no detectable effect on c-fos mRNAlevels during TPA induction (the sequences of ISIS 11184-11186 are givenin Table 2). Finally, it can also be seen from Table 5 that neitheractive oligonucleotide has any detectable effect on mRNA levels of theother active oligonucleotide's target. That is, ISIS 10639, targeted toc-fos, has no detectable effect on c-jun levels; similarly, ISIS 10582,targeted to c-jun, has no detectable effect on c-fos levels.

TABLE 5 Specificity of c-fos and c-jun Oligonucleotides Treatment c-fosC-jun Basal 5 23 TPA - no oligo 100 100 10639: c-fos active 9 97 11184:c-fos sense 84 91 11185: c-fos mismatch 93 98 11186: c-fos scrambled 9899 10582: c-jun active 91 4 11562: c-jun sense 89 87 11563: c-junscrambled 99 93 11564: c-jun mismatch 99 71

These results demonstrate that ISIS 10582 effects potent and specificmodulation (i.e., inhibition) of c-jun mRNA levels and that ISIS 10639effects potent and specific modulation of c-fos MRNA levels.

Example 4 Effect of Oligonucleotides Targeted to an AP-1 Subunit onHuman Tumor Growth in Nude Mice

In order to evaluate the in vivo activity of c-fos oligonucleotides, 25mg of tumor fragments of A549 tumors were implanted subcutaneously innude mice (n=6). ISIS 10639 was administered daily, i.v., for threeweeks. The oligonucleotide dosage was 25 mg/kg. Tumor size was recordedweekly, and the results are shown in Table 6. A substantial reduction intumor growth rate was obtained upon treatment with ISIS 10639. By day34, saline-treated tumors were 0.56 ±0.12 g by weight, while tumorstreated with ISIS 10639 were 0.31 ±0.1 g by weight.

TABLE 6 Response of Transplanted Tumors in Mice to OligonucleotidesTargeted to c-jun Mean Tumor Treatment/Time Weight (g) Std. Dev. Std.Error Saline: Day 17 0.113 0.033 0.014 Day 20 0.177 0.045 0.019 Day 270.272 0.086 0.035 Day 34 0.560 0.293 0.120 ISIS 10639: Day 17 0.1050.035 0.014 Day 20 0.138 0.074 0.030 Day 27 0.225 0.070 0.028 Day 340.310 0.104 0.042

Example 5 Effect of Oligonucleotides on Protein Levels of AP-1 Subunits

The ability of the c-fos active oligonucleotide ISIS 10639 to reduceexpression of the c-Fos protein was examined as follows. A549 cells weretreated with oligonucleotides as in Examples 2-3, except that inductionof c-Fos was effected by treatment of cells with TPA (1 μM) for threehours. At this time, whole cell protein was extracted in SDS (sodiumdodecyl sulfate) buffer. Samples of extracts were electrophoresed,transferred to nitrocellulose filters which were immunoblotted using ac-Fos-specific antibody (Santa Cruz AB, Santa Cruz, Calif.). The results(Table 7) demonstrate that treatment of cells with the c-fos antisenseoligonucleotide results in basal levels of c-Fos protein.

TABLE 7 Effect of c-fos Oligonucleotides on c-Fos Protein LevelsTreatment c-Fos Basal 21 TPA - no oligo 100 10639: c-fos active 1911184: c-fos sense 97 11185: c-fos mismatch 91 11186: c-fos scrambled 99

Example 6 Modified Oligonucleotides and PNA Antisense Analogs to HumanAP-1 Subunits

Once oligonucleotides that modulate c-Fos or c-Jun are identified,derivative or modified oligonucleotides having the same sequence thereasare prepared. In order to evaluate the effect of chemical modificationsto oligonucleotides to c-fos and c-jun, the modified oligonucleotidesdescribed in Tables 8 and 9 were prepared. The effect of thec-fos-targeted oligonucleotides on c-fos RNA levels were evaluated asdescribed in Examples 2-3. The results (Table 10) demonstrate that someenhancement of c-fos modulation can be achieved by the use modificationssuch as, e.g., 2′-fluoro (ISIS 11200). Other modified oligonucleotidesof the invention are evaluated in like fashion. In order to evaluate theeffect of PNA antisense analogs, the PNA analogs of the invention areintroduced into appropriate cell lines by microinjection according tothe method of Hanvey et al. (Science, 1992, 258:1481). Intracellulardelivery of PNA analogs is confirmed by the use of a fluorescentlytagged PNA antisense analog conjugate such as, e.g., ISIS 14240.

TABLE 8 Additional Oligonucleotides and PNA Antisense Analogs Targetedto Human AP-1 Subunits SEQ Oligonucleotide Sequence (5′->3′) ID ISIS #Target and Chemical Modifications* NO: C-JUN: 10570 & Derivatives: 10570c-jun, 5′  capG^(S)C^(S)C^(S)A^(S)C^(S)A^(S)C^(S)T^(S)C^(S)A^(S)G^(S)T^(S)G^(S)C^(S)A^(S)A^(S)C^(S)T^(S)C^(S)TP = S 1 13306 c-jun, 5′  cap    C^(S)A^(S)C^(S)T^(S)C^(S)A^(S)G^(S)T^(S)G^(S)C^(S)A^(S)A^(S)C^(S)T^(S)C^(S)TP = S 36 13297 c-jun, 5′  cap     C ^(S) A ^(S) C ^(S) T ^(S) C ^(S) A^(S) G ^(S) T ^(S) G ^(S) C ^(S) A ^(S) A ^(S) C ^(S) T ^(S) C ^(S) T P= S/2′MO 36 12166 c-jun, 5′  cap    C^(N)A^(N)C^(N)T^(N)C^(N)A^(N)G^(N)T^(N)G^(N)C^(N)A^(N)A^(N)C^(N)T^(N)C^(N)T^(N)PNA(N) 36 13699 c-jun, 5′  cap    C^(N)A^(N)C^(N)T^(K)C^(N)A^(N)G^(N)T^(K)G^(N)C^(N)A^(N)A^(N)C^(N)T^(K)C^(N)T^(K)PNA(N)/Lys(K) 36 10579 & Derivatives: 10579 c-jun, ORFC^(S)G^(S)C^(S)C^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)AP = S 5 11567 c-jun, ORFC^(S)G^(S)C^(S)C^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)A2′F 5 10571 & Derivatives: 10571 c-jun, 5′-UTRC^(S)G^(S)C^(S)A^(S)C^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)C^(S)C^(S)C^(S)G^(S)C^(S)C^(S)T^(S)CP = S 2 11568 c-jun, 5′-UTR C ^(S) G ^(S) C ^(S) A ^(S) C ^(S) C ^(S) T^(S) C ^(S) C ^(S) A ^(S) C ^(S) T ^(S) C ^(S) C ^(S) C ^(S) G ^(S) C^(S) C ^(S) T ^(S)C 2′F 2 13307 c-jun, 5′-UTR    C^(S)C^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)C^(S)C^(S)C^(S)G^(S)C^(S)C^(S)T^(S)CP = S 37 13296 c-jun, 5′-UTR     C ^(S) C ^(S) T ^(S) C ^(S) C ^(S) A^(S) C ^(S) T ^(S) C ^(S) C ^(S) C ^(S) G ^(S) C ^(S) C ^(S) T ^(S)C P =S/2′MO 37 12167 c-jun, 5′-UTR    C^(N)C^(N)T^(N)C^(N)C^(N)A^(N)C^(N)T^(N)C^(N)C^(N)C^(N)G^(N)C^(N)C^(N)T^(N)C^(N)PNA(N) 37 13698 c-jun, 5′-UTR    C^(N)C^(N)T^(K)C^(N)C^(N)A^(N)C^(N)T^(K)C^(N)C^(N)C^(N)G^(N)C^(N)C^(N)T^(K)C^(N)PNA(N)/Lys(K) 37 10582 & Derivatives: 10582 c-jun, 3′-UTRT^(S)C^(S)A^(S)G^(S)C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)A^(S)C^(S)G^(S)G^(S)T^(S)C^(S)T^(S)C^(S)T^(S)CP = S 8 11569 c-jun, 3′-UTR T ^(S) C ^(S) A ^(S) G ^(S) C ^(S) C^(S)C^(S)C^(S)C^(S)G^(S)A^(S)C^(S)G^(S)G^(S) T ^(S) C ^(S) T ^(S) C ^(S)T ^(S)C 2′F 8 11537 c-jun, 3′-UTR T ^(S) C ^(S) A ^(S) G ^(S) C ^(S) C^(S)C^(S)C^(S)C^(S)G^(S)A^(S)C^(S)G^(S)G^(S) T ^(S) C ^(S) T ^(S) C ^(S)T ^(S)C 2′propoxy- 8 14314 c-jun, 3′-UTR T ^(S) C ^(S) A ^(S) G ^(S) C^(S)C^(S)C^(S)C^(S)C^(S)G^(S)A^(S)C^(S)G^(S)G^(S)T^(S) C ^(S) T ^(S) C^(S) T ^(S)C 2′ME 8 C-FOS: 10639 & Derivatives: 10639 c-fos, 3′-UTRA^(S)A^(S)G^(S)T^(S)C^(S)C^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S)A^(S)C^(S)A^(S)G^(S)CP = S 21 11200 c-fos, 3′-UTR A ^(S) A ^(S) G ^(S) T ^(S) C ^(S) C^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C^(S)C^(S) C ^(S) A ^(S) C ^(S) A ^(S)G ^(S)C 2′MO 21 11538 c-fos, 3′-UTR A ^(S) A ^(S) G ^(S) T ^(S) C ^(S) C^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C^(S)C^(S) C ^(S) A ^(S) C ^(S) A ^(S)G ^(S)C 2′propoxy- 21 14315 c-fos, 3′-UTR A ^(S) A ^(S) G ^(S) T ^(S) C^(S)C^(S)T^(S)T^(S)G^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S) A ^(S) C ^(S) A^(S) G ^(S)C 2′ME 21 13298 & Derivatives: 13298 c-fos, ORF    T ^(S) G^(S) C ^(S) G ^(S) G ^(S) G ^(S) T ^(S) G ^(S) A ^(S) G ^(S) T ^(S) G^(S) G ^(S) T ^(S) A ^(S)G 2′ME 120 12165 c-fos, ORF   T^(N)G^(N)C^(N)G^(N)G^(N)G^(N)T^(N)G^(N)A^(N)G^(N)T^(N)G^(N)G^(N)T^(N)A^(N)G^(N)PNA(N) 120 13646 c-fos, ORF   T^(N)G^(N)C^(N)G^(N)G^(N)G^(N)T^(K)G^(N)A^(N)G^(N)T^(K)G^(N)G^(N)TKA^(N)G^(N)PNA(N)/Lys(K) 120 10628 & Derivatives: 10628 c-fos, 5′  capT^(S)G^(S)C^(S)T^(S)C^(S)G^(S)C^(S)T^(S)G^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)C^(S)G^(S)G^(S)T^(S)TP = S 10 13302 c-fos, 5′  cap   C^(S)G^(S)C^(S)T^(S)G^(S)C^(S)A^(S)G^(S)A^(S)T^(S)G^(S)C^(S)G^(S)G^(S)T^(S)TP = S 121 13301 c-fos, 5′  cap    C ^(S) G ^(S) C ^(S) T ^(S) G ^(S) C^(S) A ^(S) G ^(S) A ^(S) T ^(S) G ^(S) C ^(S) G ^(S) G ^(S) T ^(S) T P= S, 2′MO 121 12162 c-fos, 5′  cap   C^(N)G^(N)C^(N)T^(N)G^(N)C^(N)A^(N)G^(N)A^(N)T^(N)G^(N)C^(N)G^(N)G^(N)T^(N)TPNA(N) 121 13643 c-fos, 5′  cap   C^(N)G^(N)C^(N)T^(K)G^(N)C^(N)A^(N)G^(N)A^(N)T^(K)G^(N)C^(N)G^(N)G^(N)T^(K)TPNA(N)/Lys(K) 121 13303 & Derivatives: 13303 c-fos, 5′ -UTR   C^(S)C^(S)G^(S)C^(S)C^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)G^(S)T^(S)C^(S)T^(S)TP = S 122 13300 c-fos, 5′ -UTR    C ^(S) C ^(S) G ^(S) C ^(S) C ^(S) G^(S) G ^(S) C ^(S) T ^(S) C ^(S) A ^(S) G ^(S) T ^(S) C ^(S) T ^(S)T P =S, 2′MO 122 12163 c-fos, 5′ -UTR   C^(N)C^(N)G^(N)C^(N)C^(N)G^(N)G^(N)C^(N)T^(N)C^(N)A^(N)G^(N)T^(N)C^(N)T^(N)T^(N)PNA(N) 122 13644 c-fos, 5′ -UTR   C^(N)C^(N)G^(N)C^(N)C^(N)G^(N)G^(N)C^(N)T^(K)C^(N)A^(N)G^(N)T^(K)C^(N)T^(K)T^(K)PNA(N)/Lys(K) 122 13304 & Derivatives: 13304 c-fos, tIRC^(S)A^(S)T^(S)C^(S)G^(S)T^(S)G^(S)G^(S)C^(S)G^(S)G^(S)T^(S)T^(S)A^(S)G^(S)GP = S 123 13299 c-fos, tIR C ^(S) A ^(S) T ^(S) C ^(S) G ^(S) T ^(S) G^(S) G ^(S) C ^(S) G ^(S) G ^(S) T ^(S) T ^(S) A ^(S) G ^(S)G P = S,2′MO 123 12164 c-fos, tIRC^(N)A^(N)T^(N)C^(N)G^(N)T^(N)G^(N)G^(N)C^(N)G^(N)G^(N)T^(N)T^(N)A^(N)G^(N)G^(N)PNA(N) 123 13700 c-fos, tIRC^(K)A^(N)T^(K)C^(K)G^(N)T^(K)G^(N)G^(N)C^(K)G^(N)G^(N)T^(K)T^(K)A^(N)G^(N)G^(K)PNA(N)/Lys(K) 123 14240 c-fos, tIRC^(K)A^(N)T^(K)C^(K)G^(N)T^(K)G^(N)G^(N)C^(K)G^(N)G^(N)T^(K)T^(K)A^(N)G^(N)G^(K)PNA(N)/Lys(K)/5′FITC 123 *Phosphorothioate linkages are indicated by“^(S)” and “P = S”; emboldened residues indicate the additionalindicated modifications: 2′F = 2′-fluoro-; 2′propoxy = 2′-propoxy-; 2′MO= 2′methoxy-; 2′ME = 2′-methoxyethoxy-; PNA = peptide (polyamide)nucleic acid backbone having a side chain corresponding to that ofeither glycine (N) or D-Lys (K); 5′FITC = 5′-fluorescein #isothiocyanate.

TABLE 9 5-Methyl-Cytosine, Fully 2′-Methoxyethoxy- OligonucleotidesTargeted to the 3′-UTR of Human c-fos SEQ ISIS ID NO. SEQUENCE NO. 15828C^(O)C^(O)A^(O)T^(O)C^(O)T^(O)T^(O)A^(O)A^(O)T^(O)A^(O)A^(O)A^(O)T^(O)A^(O)A^(O)A^(O)T^(O)T^(O)A^(O)A^(O)A^(O)A^(O)A^(O)C^(O)A^(O)C^(O)A^(O)A^(O)T131 14660            A ^(O)A^(O) A ^(O)T^(O)A^(O) A ^(O)A^(O)T^(O)T^(O)A ^(O)A^(O) A ^(O)A^(O) A ^(O)C^(O) A ^(O)C^(O) A ^(O)A^(O)T 2,6-A* 13214659              A ^(O)A^(O)T^(O)T^(O) A ^(O)A^(O) A ^(O)A^(O) A^(O)C^(O) A ^(O)C^(O) A ^(O)A^(O)T^(O) A ^(O)A^(O) A ^(O)A^(O)C 2,6-A133 15829    A^(O)T^(O)A^(O)T^(O)A^(O)A^(O)A^(O)T^(O)A^(O)T^(O)C^(O)T^(O)G^(O)A^(O)G^(O)A^(O)A^(O)T^(O)C^(O)C134 14662     A ^(O)T^(O) A ^(O)T^(O) A ^(O)A^(O) A^(O)T^(O)A^(O)T^(O)C^(O)T^(O)G^(O)A^(O)G^(O)A^(O) A ^(O)T^(O)C^(O)C2,6-A 135 14661              A ^(O)T^(O)C^(O)T^(O)G^(O)A^(O)G^(O)A^(O) A^(O)T^(O)C^(O)C^(O) A ^(O)T^(O)C^(O)T^(O)T^(O) A ^(O) A ^(O) T 2,6-A 13614663**A^(O)A^(O)A^(O)T^(O)A^(O)T^(O)A^(O)A^(O)A^(O)T^(O)A^(O)T^(O)C^(O)T^(O)G^(O)A^(O)G^(O)A^(O)A^(O)T2,6-A 137 14664A^(O)A^(O)G^(O)A^(O)C^(O)C^(O)T^(O)C^(O)A^(O)A^(O)G^(O)G^(O)T^(O)A^(O)G^(O)A^(O)A^(O)A^(O)A^(O)A138 *Emboldened residues indicate 2,6-A residues, i.e., those having2,6-diaminopurine as a nucleobase. **ISIS 14663 is a 2′-deoxy- ratherthan a 2′-methoxyethoxy-oligonucleotide.

TABLE 10 Effect of Modified c-fos Oligonucleotides on c-fos ExpressionTreatment c-fos Basal 3 TPA - no oligo 100 10639: c-fos active, P = S 911200: c-fos, 2′-fluoro- 5 11538: c-fos, 2′-propoxy- 15

Example 7 Oligonucleotides to Mouse AP-1 Subunits

Tables 11 and 12 show the sequences of oligonucleotides designed tomodulate mouse c-jun and c-fos mRNA expression, respectively. For thenucleotide sequence of the mouse c-jun gene, see Genbank accession No.J04115/MUSCJUN and Ryder et al., Proc. Natl. Acad. Sci. U.S.A., 1988,85:8464. The nucleotide sequence of the mouse c-fos gene is described inGenbank accession No. J00370/MUSFOS and by Van Beveren et al., Cell,1983, 32:1241. Oligonucleotide activities are evaluated as describedinfra in Example 2 et seq. with the exception that mouse Swiss 3T3 cells(available from, inter alia, the American Type Culture Collection,Rockville, Md., as ATCC No. CCL-92) are used instead of human A549cells. Due to the high degree of homology between human and murine c-junand c-fos nucleotide sequences (Van Straaten et al., Proc. Natl. Acad.Sci. U.S.A., 1983, 80:3183), probes derived from the human genes wereused to detect murine messages.

TABLE 11 Phosphorothioate Oligonucleotides Targeted to Mouse c-jun ISIS# SEQUENCE SEQ ID NO: TARGET REGION* 12292 CTC-GCC-CAA-CTT-CAG-CCG-CC 385′-UTR: 434-453 12293 CCA-GTC-CCA-GCA-ACA-GCG-GC 39 5′-UTR: 588-60712294 GCA-ACA-GCG-CGC-CGG-GAA-GC 40 5′-UTR: 838-857 12295CCG-GCG-ACG-CCA-GCT-TGA-GC 41 ORF: 1120-1139 12296GGC-TGT-GCC-GCG-GAG-GTG-AC 42 ORF: 1304-1323 12297CGC-CCC-ACC-GCC-GCT-GCT-CA 43 ORF: 1458-1477 12298AGC-CCG-GCC-GCG-CCA-TAG-GA 44 ORF: 1481-1500 12299CTG-CAC-CGG-GAT-CTG-TTG-GG 45 ORF: 1560-1579 12300GGC-GGC-GTC-TCT-CCC-GGC-ATC-TC 46 ORF: 1625-1647 12301TGG-AGG-CGG-CAA-TGC-GGT-TC 47 ORF: 1708-1727 12302CCC-TGA-GCA-TGT-TGG-CCG-TG 48 ORF: 1813-1832 12303CAA-AGC-CAG-GCG-CGC-CAC-GT 49 3′-UTR: 2096-2115 12304TTG-AGA-GAG-GCA-GGC-CAG-GG 50 3′-UTR: 2388-2407 12305TGG-ACT-TGT-GTG-TTG-CCG-GG 51 3′-UTR: 2807-2826 12306TCC-ATG-GGT-CCC-TGC-TTT-GA 52 3′-UTR: 2999-3018 12321TGG-TCG-CGC-GCG-GGC-ACA-GC 53 3′-UTR: 2166-2185 *Nucleotide co-ordinatesfrom Genbank accession No. J04115/MUSCJUN.

TABLE 12 Phosphorothioate Oligonucleotides Targeted to Mouse c-fos ISIS# SEQUENCE SEQ ID NO: TARGET REGION* 11249 AGC-TCC-CTC-CTC-CGA-TTC-CG 54ORF: 1905-1924 11250 GCT-CTG-TGA-CCA-TGG-GCC-CC 55 ORF: 2498-2517 12307GAA-CCG-CCG-GCT-CTA-TCC-AG 56 5′-UTR: 164-183 12308GCC-CCT-GCG-AGT-CAC-ACC-CC 57 ORF: 485-504 12309TAA-GGC-TGC-TCT-GAC-CGC-GC 58 ORF: 541-560 12310CGC-CCG-CAG-CAC-CCT-CCT-CC 59 ORF: 804-823 12311CAG-GCG-CTG-CTC-CGG-AGT-CT 60 ORF: 868-887 12312TCC-CTT-GAA-TTC-CGC-AGC-GC 61 ORF: 989-1008 12313AGC-GGA-GGT-GAG-CGA-GGA-GG 62 ORF: 1136-1155 12314CCC-CAG-CCC-ACA-AAG-GTC-CA 63 ORF: 1445-1464 12315TGC-TCA-AGG-ACC-CTG-CGC-CC 64 ORF: 2001-2020 12316GGG-AAG-CCA-AGG-TCA-TCG-GG 65 ORF: 2178-2197 12317TGC-TGC-TGC-CCT-TTC-GGT-GG 66 ORF: 2630-2649 12318CTG-GAT-GCC-GGC-TGC-CTT-GC 67 3′-UTR: 2716-2735 12319CAG-CTC-GGG-CAG-TGG-CAC-GT 68 3′-UTR: 2736-2755 12320GGA-ACA-CGC-TAT-TGC-CAG-GA 69 3′-UTR: 3138-3157 *Nucleotide co-ordinatesfrom Genbank accession No. J00370/MUSFOS.

Example 8 Oligonucleotides to Rat AP-1 Subunits

Tables 13 and 14 show the sequences of oligonucleotides designed tomodulate rat c-jun and c-fos mRNA expression, respectively. For thenucleotide sequence of the rat c-jun gene, see Genbank accession No.X17163/RSJUNAP1 and Saaki et al., Cancer Res., 1989, 49:5633. Thenucleotide sequence of the rat c-fos gene is described in Genbankaccession No. X06769/RNCFOSR and Curran et al., Oncogene, 1987, 2:79.Oligonucleotide activities were evaluated as described infra in Example2 et seq. with the exception that rat A10 cells (available from, interalia, the American Type Culture Collection, Rockville, Md., as ATCC No.CRL-1476) were used instead of human A549 cells. Due to the high degreeof homology between human and rat c-jun and c-fos nucleotide sequences,probes derived from the human genes were used to detect the ratmessages.

ISIS 12633 (SEQ ID NO:78), a 20-mer phosphorothioate oligonucleotidecomplementary to a portion of the 3′ UTR of rat c-jun mRNA, was selectedas an active modulator of c-jun for further studies. Another preferredoligonucleotide targeted to rat AP-1 subunits is ISIS 12635 (SEQ IDNO:80).

Example 9 Modified Oligonucleotides to Rat AP-1 Subunits

Tables 15 and 16 show the sequences and chemical modifications of secondgeneration oligonucleotides designed to modulate mouse c-jun and c-fosmRNA expression. The activities of these modified oligonucleotide areevaluated as described infra in Example 8.

TABLE 13 Phosphorothioate Oligonucleotides Targeted to Rat c-jun ISIS #SEQUENCE SEQ ID NO: TARGET REGION* 12624 CGG-CGG-CGC-AGA-CCA-GTC-GT 705′-UTR: 2-21 12625 GCC-GCG-GGA-CCA-GCC-CCA-GC 119 5′-UTR: 35-54 12626GGC-ATC-GTC-GTA-GAA-GGT-CG 71 5′-UTR: 20-39 12627GGA-GGT-GCG-GCT-TCA-GAT-TG 72 ORF: 493-512 12628CCC-TCC-TGC-TCG-TCG-GTC-AC 73 5′-UTR: 307-326 12629ACT-GAC-TGG-TTG-TGC-CGC-GG 74 ORF: 747-766 12630CGC-TGT-AGC-CGC-CGC-CGC-CG 75 ORF: 814-833 12631CCT-TGA-TCC-GCT-CCT-GAG-AC 76 ORF: 1105-1124 12632GCC-AGC-TCG-GAG-TTT-TGC-GC 77 ORF: 1226-1245 12633TTT-TCT-TCC-ACT-GCC-CCT-CA 78 3′-UTR: 1375-1394 12634CCC-TTG-GCT-TCA-GTA-CTC-GG 79 3′-UTR: 1451-1470 12635CTT-CCC-ACT-CCA-GCA-CAT-TG 80 3′-UTR: 1509-1528 12636GCA-CAG-CCC-GTT-CGC-AAA-GC 81 3′-UTR: 1584-1603 12637AAT-GCA-GCA-GAG-AGG-TTG-GG 82 3′-UTR: 2089-2108 12638GAC-GGG-AGG-GAC-TAC-AGG-CT 83 3′-UTR: 2168-2187 12639TCT-GGA-CTT-GTG-GGT-TGC-TG 84 3′-UTR: 2240-2259 12640TAA-ACG-ATC-ACA-GCG-CAT-GC 85 3′-UTR: 2375-2394 12628 controls: 12628CCC-TCC-TGC-TCG-TCG-GTC-AC 73 5′-UTR 12893 GGG-AGG-ACG-AGC-AGC-CAG-TG 86reverse sense control 12894 CCC-GGC-CTT-TTG-ACC-GCC-TC 87 scrambledcontrol 12895 CCG-CCT-CCC-CGG-CCT-TTT-GA 88 scrambled control 12896CCG-TCG-TGG-TCC-TCC-GTG-AC 89 mismatch control 12992GTG-ACC-GAC-GAG-CAG-GAG-GG 90 sense control 12633 controls: 12633TTT-TCT-TCC-ACT-GCC-CCT-CA 78 3′-UTR 12897 AAA-AGA-AGG-TGA-CGG-GGA-GT 91reverse sense control 12898 TTC-TCT-TTT-AGC-CTC-CCC-CA 92 scrambledcontrol 12899 TCC-CCC-ATT-CTC-TTT-TAG-CC 93 scrambled control 12900TTA-TCA-TCG-ACA-GCG-CCA-CA 94 mismatch control 12993TGA-GGG-GCA-GTG-GAA-GAA-AA 95 sense control *Nucleotide co-ordinatesfrom Genbank accession No. X17163/RSJUNAP1.

TABLE 14 Phosphorothioate Oligonucleotides Targeted to Rat c-fos ISIS #SEQUENCE SEQ ID NO: TARGET REGION* 11124 GTT-CTC-GGC-TCC-GCC-GGC-TC 965′-UTR: 22- 41 11245 CAT-CAT-GGT-CGT-GGT-TTG-GG 97 tIR: 122-140 12246TCC-GCG-TTG-AAA-CCC-GAG-AA 98 ORF: 141-160 12247TGG-GCT-GGT-GGA-GAT-GGC-TG 99 ORF: 328-347 12248CGA-TGC-TCT-GCG-CTC-TGC-CG 100 ORF: 485-504 12251TTC-GGT-GGG-CAG-CTG-CGC-AG 101 ORF: 1193-1212 12252CAG-GGC-TAG-CAG-TGT-GGG-CG 102 tTR: 1255-1274 12253CCA-GCT-CAG-TCA-GTG-CCG-GC 103 3′-UTR: 1299-1318 12254TCT-ACG-GGA-ACC-CCT-CGA-GG 104 3′-UTR: 1348-1367 12255CTC-CAT-GCG-GTT-GCT-TTT-GA 105 3′-UTR: 1518-1537 12256CAG-GCC-TGG-CTC-ACA-TGC-TA 106 3′-UTR: 1576-1595 11254 controls: 11254TCT-ACG-GGA-ACC-CCT-CGA-GG 104 3′-UTR 12698 AGA-TGC-CCT-TGG-GGA-GCT-CC107 reverse sense control 12699 TGA-CTA-TAG-ACC-GCC-GCC-GG 108 scrambledcontrol 12700 CCG-CCG-GTG-ACT-ATA-GAC-CG 109 scrambled control 12728TCA-ACC-GGT-ACG-CCA-CGT-GG 110 mismatch control 12990CCT-CGA-GGG-GTT-CCC-GTA-GA 111 sense control 11256 controls: 11256CAG-GCC-TGG-CTC-ACA-TGC-TA 106 12701 GTC-CGG-ACC-GAG-TGT-ACG-AT 112reverse sense control 12702 GCG-CAC-CGT-CAT-TAC-GTC-GA 113 scrambledcontrol 12703 ACG-TCG-AGC-GCA-CCG-TCA-TT 114 scrambled control 12729CAC-GCG-TGC-CTG-ACT-TGG-TA 115 mismatch control 12991TAG-CAT-GTG-AGC-CAG-GCC-TG 116 sense control *Nucleotide co-ordinatesfrom Genbank accession No. X06769/RNCFOSR (see also Curran et al.,Oncogene, 198, 2:79).

TABLE 15 Additional Oligonucleotides Targeted to Rat c-junOligonucleotide Sequence (5′->3′) SEQ ID ISIS # Target and ChemicalModifications* NO: 12633 & Derivatives: 12633 3′-UTRT^(S)T^(S)T^(S)T^(S)C^(S)T^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(S)C^(S)C^(S)T^(S)C^(S)A48 13047 3′-UTR T ^(O) T ^(O) T ^(O) T ^(O) C ^(O) T^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S) C ^(O) C ^(O) C ^(O) T ^(O)C ^(O) A ^(d) 2′MO 78 13714 3′-UTR T ^(O) T ^(O) T ^(O) T ^(O) C ^(O) T^(S)T^(S)C^(S)C^(S)A^(S)C^(S)T^(S)G^(S)C^(S)C^(O)C^(O)C^(O)T^(O)C^(O)A^(d)2′ME 78 15707 3′-UTR T ^(S) T ^(S) T ^(S) T^(S)C^(O)T^(O)T^(O)C^(O)C^(O)A^(O)C^(O)T^(O)G^(O)C^(O)C^(O)C^(S)C^(S)T^(S)C^(S)A^(d)2′ME 78 15708 3′-UTR T ^(S) T ^(S) T ^(S) T ^(S) C ^(S) T^(O)T^(O)C^(O)C^(O)A^(O)C^(O)T^(O)G^(O)C^(O)C^(S) C ^(S) C ^(S) T ^(S) C^(S) A ^(d) 2′ME 78 13881 3′-UTR T ^(O) T ^(O) T ^(O) T ^(O) C ^(O) T^(O) T ^(O) C ^(O) C ^(O) A ^(O) C ^(O) T^(O)G^(S)C^(S)C^(S)C^(S)C^(S)T^(S)C^(S)A^(d) 2′ME 78 13882 3′-UTRT^(S)T^(S)T^(S)T^(S)C^(S)T^(S)T^(S)C^(S) C ^(O) A ^(O) C ^(O) T ^(O) G^(O) C ^(O) C ^(O) C ^(O) C ^(O) T ^(O) C ^(O)A^(d) 2′ME 78 12898(scrambled 12633) & Derivatives: 12898 controlT^(S)T^(S)C^(S)T^(S)C^(S)T^(S)T^(S)T^(S)T^(S)A^(S)G^(S)C^(S)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)C^(S)A92 13046 control T ^(O) T ^(O) C ^(O) T ^(O) C ^(O) T^(S)T^(S)T^(S)T^(S)A^(S)G^(S)C^(S)C^(S)T^(S)C^(O)C^(O)C^(O)C^(O)C^(O)A^(d)2′MO 92 13715 control T ^(O) T ^(O) C ^(O) T ^(O) C ^(O) T^(S)T^(S)T^(S)T^(S)A^(S)G^(S)C^(S)C^(S)T^(S)C^(O)C^(O)C^(O)C^(O)C^(O)A^(d)2′ME 92 13912 control T ^(O) T ^(O) C ^(O) T ^(O) C ^(O) T ^(O) T^(O)T^(O) T ^(O) A ^(O) G ^(O) C^(O)C^(O)C^(S)T^(S)C^(S)C^(S)C^(S)C^(S)A^(d) 2′ME 92 13913 controlT^(S)T^(S)C^(S)T^(S)C^(S)T^(S)T^(S)T^(S) T ^(O) A ^(O) G^(O)C^(O)C^(O)T^(O)C^(O)C^(O)C^(O)C^(O)C^(O)A^(d) 2′ME 92 15705 controlT ^(S) T ^(S) C ^(S) T^(S)C^(O)T^(O)T^(O)T^(O)T^(O)A^(O)G^(O)C^(O)C^(O)T^(O)C^(O)C^(S) C ^(S)C ^(S) C ^(S) A ^(d) 2′ME 92 15706 control T ^(S) T ^(S) C ^(S) T^(S)C^(O)T^(O)T^(O)T^(O)T^(O)A^(O)G^(O)C^(O)C^(O)T^(O)C^(O)C^(O)C^(S) C^(S) C ^(S) A ^(d) 2′ME 92 12628 & Derivatives: 12628 5′-UTRC^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S)G^(S)G^(S)T^(S)C^(S)A^(S)C73 13049 5′-UTRC^(O)C^(O)C^(O)T^(O)C^(O)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S) G^(O) G ^(O) T ^(O) C ^(O) A ^(O)C^(d) 2′MO 73 13712 5′-UTRC^(O)C^(O)C^(O)T^(O)C^(O)C^(S)T^(S)G^(S)C^(S)T^(S)C^(S)G^(S)T^(S)C^(S) G^(O) G ^(O) T ^(O) C ^(O) A ^(O)C^(d) 2′ME 73 13879 5′-UTR C ^(O) C ^(O)C ^(O) T ^(O) C ^(O) C ^(O) T ^(O) G ^(O) C ^(O) T^(O)C^(O)G^(S)T^(S)C^(S)G^(S)G^(S)T^(S)C^(S)A^(S)C^(d) 2′ME 73 138805′-UTR C^(S)C^(S)C^(S)T^(S)C^(S)C^(S)T^(S)G^(S) C ^(O) T ^(O) C ^(O) G^(O) T ^(O) C ^(O) G ^(O) G ^(O) T ^(O) C ^(O) A ^(O) C ^(d) 2′ME 7312894 (scrambled 12628) & Derivatives: 12894C^(S)C^(S)C^(S)G^(S)G^(S)C^(S)C^(S)T^(S)T^(S)T^(S)T^(S)G^(S)A^(S)C^(S)C^(S)G^(S)C^(S)C^(S)T^(S)C87 13713 C ^(O) C ^(O) C ^(O) G ^(O) G ^(O) C^(S)C^(S)T^(S)T^(S)T^(S)T^(S)G^(S)A^(S)C^(S)C^(O) G ^(O) C ^(O) C ^(O) T^(O)C^(d) 2′ME 87 13048 C ^(O) C ^(O) C ^(O) G ^(O) G^(O)C^(S)C^(S)T^(S)T^(S)T^(S)T^(S)G^(S)A^(S)C^(S)C^(O) G ^(O) C ^(O) C^(O) T ^(O)C^(d) 2′MO 87 12635 & Derivative^(s): 12635C^(S)T^(S)T^(S)C^(S)C^(S)C^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C^(S)A^(S)T^(S)T^(S)G80 15711 C ^(S) T ^(S) T^(S)C^(S)C^(S)C^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C^(S) A^(S) T ^(S) T ^(S) G 2′ME 80 15712 C ^(S) T ^(S) T^(S)C^(S)C^(S)C^(S)A^(S)C^(S)T^(S)C^(S)C^(S)A^(S)G^(S)C^(S)A^(S)C^(S) A^(S) T ^(S) T ^(S) G ^(d) 2′ME 80 15709 (scrambled 12635) & Derivatives:15709 T ^(S) T ^(S) C ^(S) T^(S)C^(S)A^(S)C^(S)C^(S)C^(S)A^(S)C^(S)C^(S)A^(S)C^(S)G^(S) T ^(S) A^(S) C ^(S) G ^(S) T 2′ME 117 15710 T ^(S) T ^(S) C ^(S) T^(S)C^(O)A^(O)C^(O)C^(O)C^(O)A^(O)C^(O)C^(O)A^(O)C^(O)G^(O)T^(S) A ^(S)C ^(S) G ^(S) T 2′ME 117 *Phosphorothioate linkages are indicated by“^(S)”, whereas phosphodiester linkages are signified by “^(O)”;emboldened residues comprise the additional indicated modifications:2′MO = 2′-methoxy-; 2′ME = 2′-methoxyethoxy-.

TABLE 16 Additional Oligonucleotides Targeted to Rat c-fosOligonucleotide Sequence (5′->3′) SEQ ID ISIS # Target and ChemicalModifications* NO: 11256 & Derivatives: 106 11256 3′-UTRC^(S)A^(S)G^(S)G^(S)C^(S)C^(S)T^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)C^(S)A^(S)T^(S)G^(S)C^(S)T^(S)A106 13051 3′-UTR C ^(O) A ^(O) G ^(O) G ^(O) C ^(O) C^(S)T^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)C^(S) A ^(O) T ^(O) G ^(O) C ^(O)T ^(O)A^(d) 2′MO 106 13718 3′-UTR C ^(S) A ^(S) G ^(S) G ^(S) C ^(S) C^(S)T^(S)G^(S)G^(S)C^(S)T^(S)C^(S)A^(S)C^(S) A ^(S) T ^(S) G ^(S) C ^(S)T ^(S)A^(d) 2′ME 106 13877 3′-UTR C ^(O) A ^(O) G ^(O) G ^(O) C ^(O) C^(O) T ^(O) G^(O)G^(O)C^(O)T^(O)C^(O)A^(S)C^(S)A^(S)T^(S)G^(S)C^(S)T^(S)A^(d) 2′ME106 13878 3′-UTRC^(O)A^(O)G^(O)G^(O)C^(O)C^(O)T^(O)G^(O)G^(S)C^(S)T^(S)C^(S) A ^(S) C^(S) A ^(S) T ^(S) G ^(S) C ^(S) T ^(S)A^(d) 2′ME 106 12703 (Scrambled11256) & Derivatives: 12703 controlA^(S)C^(S)G^(S)T^(S)C^(S)G^(S)A^(S)G^(S)C^(S)G^(S)C^(S)A^(S)C^(S)C^(S)G^(S)T^(S)C^(S)A^(S)T^(S)T114 13050 control A ^(O) C ^(O) G ^(O) T ^(O) C ^(O) G^(S)A^(S)G^(S)C^(S)G^(S)C^(S)A^(S)C^(S)C^(S) G ^(O) T ^(O) C ^(O) A ^(O)T ^(O)T^(d) 2′MO 114 13719 control A ^(S) C ^(S) G ^(S) T ^(S) C ^(S) G^(S)A^(S)G^(S)C^(S)G^(S)C^(S)A^(S)C^(S)C^(S) G ^(S) T ^(S) C ^(S) A ^(S)T ^(S)T^(d) 2′ME 114 11254 & Derivatives: 11254 3′-UTRT^(S)C^(S)T^(S)A^(S)C^(S)G^(S)G^(S)G^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)T^(S)C^(S)G^(S)A^(S)G^(S)G104 13053 3′-UTR T ^(O) C ^(O) T ^(O) A^(O)C^(O)G^(S)G^(S)G^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S) T ^(O) C ^(O) G^(O) A ^(O) G ^(O)G^(d) 2′MO 104 13716 3′-UTR T ^(S) C ^(S) T ^(S) A^(S)C^(S)G^(S)G^(S)G^(S)A^(S)A^(S)C^(S)C^(S)C^(S)C^(S)T^(S)C^(S) G ^(S)A ^(S) G ^(S)G^(d) 2′ME 104 13875 3′-UTR T ^(O) C ^(O) T ^(O) A ^(O) C^(O) G ^(O) G ^(O) G ^(O) A ^(O) A^(O)C^(O)C^(O)C^(S)C^(S)T^(S)C^(S)G^(S)A^(S)G^(S)G^(d) 2′ME 104 138763′-UTR T^(S)C^(S)T^(S)A^(S)C^(S)G^(S)G^(S)G^(S) A ^(O) A^(O)C^(O)C^(O)C^(O)C^(O)T^(O)C^(O) G ^(O) A ^(O) G ^(O)G^(d) 2′ME 10412700 (Scrambled 11254) & Derivatives: 12700 controlC^(S)C^(S)G^(S)C^(S)C^(S)G^(S)G^(S)T^(S)G^(S)A^(S)C^(S)T^(S)A^(S)T^(S)A^(S)G^(S)A^(S)C^(S)C^(S)G109 13052 controlC^(O)C^(O)G^(O)C^(O)C^(O)G^(S)G^(S)T^(S)G^(S)A^(S)C^(S)T^(S)A^(S)T^(S) A^(O) G ^(O) A ^(O)C^(O)C^(O)G^(d) 2′MO 109 13717 controlC^(S)C^(S)G^(S)C^(S)C^(S)G^(S)G^(S)T^(S)G^(S)A^(S)C^(S)T^(S)A^(S)T^(S) A^(S) G ^(S) A ^(S)C^(S)C^(S)G^(d) 2′ME 109 *Phosphorothioate andphosphodiester linkages are indicated by “^(S)” and “^(O)” respectively,whereas “^(d)” indicates a dideoxy (chain-terminating) residue;emboldened residues comprise the additional indicated modifications:2′MO, 2′-methoxy; 2′ME, 2′-methoxyethoxy-.

Example 11 Effect of Oligonucleotides Targeted to AP-1 Subunits onPDGF-Induced Proliferation of Rat Aortic Smooth Muscle Cells

In order to evaluate the effect of AP-1 modulation on cell cycleprogression, the following study was performed. Cultured rat aorticsmooth muscle (PASM) cells are stimulated to proliferate upon contactwith platelet-derived growth factor (PDGF). Primary RASM cells (passages6-8) were synchronized by incubation for 48 hours in DMEM containing0.1% FBS. The cells were treated for 4 hours with 200 nM ISIS 12633 (SEQID NO:713), a 20-mer phosphorothioate oligonucleotide complementary to aportion of the 3′ UTR of rat c-jun mRNA, or ISIS 12898 (SEQ ID NO:92), ascrambled control of ISIS 12633. Cells were then contacted with PDGF (10ng/ml) (R&D Systems, Minneapolis, Minn.), and cell cycle progression wasassessed by FACS analysis 24 hours later. At 2 and 6 hours afterexposure to PDGF, c-jun mRNA levels were markedly less in ISIS12633-treated cells as compared to untreated cells or cells treated withISIS 12898. The decrease in c-jun mRNA levels was associated with asignificant decrease in the proportion of cells in the G2/M interface at24 hours. This result provides evidence of the role of AP-1-mediatedgene expression in cellular proliferation and indicate that cell cycleprogression can be modulated by preventing expression of one or both ofthe genes which encode a subunit of AP-1.

Example 12 Effect of Oligonucleotides Targeted to AP-1 Subunits onEnzymes Involved in Metastasis

Patients having benign tumors, and primary malignant tumors that havebeen detected early in the course of their development, may often besuccessfully treated by the surgical removal of the benign or primarytumor. If unchecked, however, cells from malignant tumors are spreadthroughout a patient's body through the processes of invasion andmetastasis. Invasion refers to the ability of cancer cells to detachfrom a primary site of attachment and penetrate, e.g., an underlyingbasement membrane. Metastasis indicates a sequence of events wherein (1)a cancer cell detaches from its extracellular matrices, (2) the detachedcancer cell migrates to another portion of the patient's body, often viathe circulatory system, and (3) attaches to a distal and inappropriateextracellular matrix, thereby created a focus from which a secondarytumor can arise. Normal cells do not possess the ability to invade ormetastasize and/or undergo apoptosis (programmed cell death) if suchevents occur (Ruoslahti, Sci. Amer., 1996, 275, 72).

The matrix metalloproteinases (MMPs) are a family of enzymes which havethe ability to degrade components of the extracellular matrix(Birkedal-Hansen, Current Op. Biol., 1995, 7, 728). Many members of theMMP family have been found to have elevated levels of activity in humantumors as well as other disease states (Stetler-Stevenson et al., Annu.Rev. Cell Biol., 1993, 9, 541; Bernhard et al., Proc. Natl. Acad. Sci.(U.S.A.), 1994, 91, 4293). In particular, one member of this family,matrix metalloproteinase-9 (MMP-9), is often found to be expressed onlyin tumors and other diseased tissues (Himelstein et al., Invasion &Metastasis, 1994, 14, 246). Several studies have shown that regulationof the MMP-9 gene may be controlled by the AP-1 transcription factor(Kerr et al., Science, 1988, 242, 1242; Kerr et al., Cell, 1990, 61,267; Gum et al., J. Biol. Chem., 1996, 271, 10672; Hua et al., CancerRes., 1996, 56, 5279). In order to determine whether MMP-9 expressioncan be influenced by AP-1 modulation, the following experiments wereconducted on normal human epidermal keratinocytes (NHEKs). AlthoughNHEKs normally express no detectable MMP-9, MMP-9 can be induced by anumber of stimuli, including TPA. ISIS 10582, an oligonucleotidetargeted to c-jun, was evaluated for its ability to modulate MMP-9expression according to the protocols described in Examples 2-3 with thefollowing exceptions: (1) NHEK cells were used instead of A549 cells,(2) the probe used, a PCR product prepared using the published sequenceof the MMP-9 gene (Huhtala et al., J. Biol. Chem., 1991, 266:16485; Satoet al., Oncogene, 1993, 8:395), is specific for MMP-9 rather than c-jun,and (3) the cells were harvested 24 hours after TPA treatment for 4hours. The results (Table 17) demonstrate that ISIS 10582 is able tocompletely inhibit the expression of MMP-9 after induction with TPA.

TABLE 17 Effect of c-jun Oligonucleotide on MMP-9 Expression TreatmentMMP-9 Basal 4 TPA - no oligo 100 10582: c-jun active 6 11562: sensecontrol 99 11563: scrambled control 95 11564: mismatch control 89

These results demonstrate that c-Jun is required for TPA-mediatedinduction of MMP-9, and indicate that oligonucleotides targeted to AP-1subunits can inhibit the expression of MMP family members, therebymodulating the ability of cancer cells to invade other tissues and/ormetastasize to other sites in a patient's body.

Example 13 Antagonism of Inducers Other than TPA by OligonucleotidesTargeted to AP-1 Subunits

Inducing agents other than TPA function to raise AP-1 levels in vivo. Inorder to assess the ability of oligonucleotides targeted to AP-1 toantagonize the action of three such inducers, A549 cells were treatedand evaluated as in Examples 2 et seq. with the exception that TNF-α,IL-1β or TGF-β (each at 10 ng/ml and all from R&D Systems, Minneapolis,Minn.) were used in place of TPA as inducers. The results (Table 18)demonstrate that ISIS 10582 (SEQ ID NO:8, targeted to huran c-jun)effectively reduces stimulation of c-Jun by TNF-α or IL-1β. In contrast,a scrambled control oligonucleotide, ISIS 11563 (SEQ ID NO:30), did notreverse the induction of c-Jun.

TABLE 18 Effect of Oligonucleotides Targeted to c-Jun on Induction byTNF-α, IL-1β or TGF-β ISIS ISIS Inducer Basal No Oligo 10582 11563 TNF-α5 100 20 98 IL-1β 9 100 15 94 TGF-β 2 100 95 99

TABLE 18 Effect of Oligonucleotides Targeted to c-Jun on Induction byTNF-α, IL-1β or TGF-β ISIS ISIS Inducer Basal No Oligo 10582 11563 TNF-α5 100 20 98 IL-1β 9 100 15 94 TGF-β 2 100 95 99

What is claimed is:
 1. An antisense oligonucleotide comprising 8-30nucleobases connected by covalent linkages, wherein said antisenseoligonucleotide comprises at least an 8 nucleobase portion of SEQ ID NO:10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 120,121, 122, 123, 131, 132, 133, 134, 135, 136, 137, or 138, wherein saidantisense oligonucleotide inhibits the expression of a c-Fos protein,wherein said antisense oligonucleotide comprises at least one 2′methoxyethoxy residue, and wherein said antisense oligonucleotidecomprises at least one lipophilic moiety which enhances the cellularuptake of said antisense oligonucleotide.
 2. The antisenseoligonucleotide of claim 1, wherein said lipophilic moiety is selectedfrom the group consisting of a cholesterol moiety, a cholesteryl moiety,cholic acid, a thioether, a thiocholesterol, an aliphatic chain, aphospholipid, a polyamine chain, a polyethylene glycol chain, adamantaneacetic acid, a palmityl moiety, an octadecylamine moiety and ahexylamino-carbonyl-oxycholesterol moiety.
 3. The antisenseoligonucleotide of claim 1, wherein at least one of said linkages isselected from the group consisting of a phosphorothioate linkage, aphosphodiester linkage, a phosphotriester linkage, a methyl phosphonatelinkage, a methylene(methylimino) linkage, a morpholino linkage, anamide linkage, a polyamide linkage, a short chain alkyl intersugarlinkage, a cycloalkyl intersugar linkage, a short chain heteroatomicintersugar linkage and a heterocyclic intersugar linkage.
 4. Theantisense oligonucleotide of claim 1, wherein at least one of saidnucleotides has a modified sugar moiety.
 5. The antisenseoligonucleotide of claim 4, wherein said modified sugar moiety is amodification at the 2′ position of any nucleotide, the 3′ position ofthe 3′ terminal nucleotide or the 5′ position of the 5′ terminaloligonucleotide.
 6. The antisense oligonucleotide of claim 5, whereinsaid modification is selected from the group consisting of asubstitution of an azido group for a 3′ hydroxyl group and asubstitution of a hydrogen atom for a 3′ or 5′ hydroxyl group.
 7. Theantisense oligonucleotide of claim 5, wherein said modification is asubstitution or addition at the 2′ position of a moiety selected fromthe group consisting of —OH, —SH, —SCH₃, —F, —OCN, —OCH₃OCH₃,—OCH₃O(CH₂)_(n)CH₃, —O(CH₂)_(n)NH₂ or —O(CH₂)_(n)CH₃ where n is from 1to about 10, a C₁ to C₁₀ lower alkyl group, an alkoxyalkoxy group, asubstituted lower alkyl group, a substituted alkaryl group, asubstituted aralkyl group, —Cl, —Br, —CN, —CF₃, —OCF₃, an -O-alkylgroup, an —S-alkyl group, an N-alkyl group, an O-alkenyl group, anS-alkenyl group, an N-alkenyl group, —SOCH₃, —SO₂CH₃, —ONO₂, —NO₂, —N₃,—NH₂, a heterocycloalkyl group, a heterocycloalkaryl group, anaminoalkylamino group, a polyalkylamino group, a substituted silylgroup, an RNA cleaving group, a reporter group, a DNA intercalatinggroup, a group for improving the pharmacokinetic properties of anoligonucleotide, a group for improving the pharmacodynamic properties ofan oligonucleotide, a methoxyethoxy group and a methoxy group.
 8. Theantisense oligonucleotide of claim 1, wherein at least one of saidnucleotides has a modified nucleobase.
 9. The antisense oligonucleotideof claim 8, wherein said modified nucleobase is selected from the groupconsisting of 5 hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine,glycosyl 5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,N⁶-(6-aminohexyl)adenine, 8-azaguanine, 7-deazaguanine and2,6-diaminopurine.
 10. The antisense oligonucleotide of claim 1, whereinsaid c-Fos protein is human c-Fos.
 11. A pharmaceutical compositioncomprising the antisense oligonucleotide of claim 1, and apharmaceutically acceptable carrier.
 12. An antisense oligonucleotidecomprising 8-30 nucleotides connected by covalent linkages, wherein saidantisense oligonucleotide comprises at least an 8 nucleobase portion ofSEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 36, or 37, wherein said antisenseoligonucleotide inhibits the expression of a c-Jun protein, wherein saidantisense oligonucleotide comprises at least one 2′ methoxyethoxyresidue, and wherein said antisense oligonucleotide comprises at leastone lipophilic moiety which enhances the cellular uptake of saidantisense oligonucleotide.
 13. The antisense oligonucleotide of claim12, wherein said lipophilic moiety is selected from the group consistingof a cholesterol moiety, a cholesteryl moiety, cholic acid, a thioether,a thiocholesterol, an aliphatic chain, a phospholipid, a polyaminechain, a polyethylene glycol chain, adamantane acetic acid, a palmitylmoiety, an octadecylamine moiety and ahexylamino-carbonyl-oxycholesterol moiety.
 14. The antisenseoligonucleotide of claim 12, wherein at least one of said linkages isselected from the group consisting of a phosphorothioate linkage, aphosphodiester linkage, a phosphotriester linkage, a methyl phosphonatelinkage, a methylene(methylimino) linkage, a morpholino linkage, anamide linkage, a polyamide linkage, a short chain alkyl intersugarlinkage, a cycloalkyl intersugar linkage, a short chain heteroatomicintersugar linkage and a heterocyclic intersugar linkage.
 15. Theantisense oligonucleotide of claim 12, wherein at least one of saidnucleotides has a modified sugar moiety.
 16. The antisenseoligonucleotide of claim 15, wherein said modified sugar moiety is amodification at the 2′ position of any nucleotide, the 3′ position ofthe 3′ terminal nucleotide or the 5′ position of the 5′ terminaloligonucleotide.
 17. The antisense oligonucleotide of claim 16, whereinsaid modification is selected from the group consisting of asubstitution of an azido group for a 3′ hydroxyl group and asubstitution of a hydrogen atom for a 3′ or 5′ hydroxyl group.
 18. Theantisense oligonucleotide of claim 16, wherein said modification is asubstitution or addition at the 2′ position of a moiety selected fromthe group consisting of —OH, —SH, —SCH₃, —F, —OCN, —OCH₃OCH₃, —OCH₃O(CH₂)_(n)CH₃, —O(CH₂)_(n)NH₂ or —O(CH₂)_(n)CH₃ where n is from 1 toabout 10, a C₁ to C₁₀ lower alkyl group, an alkoxyalkoxy group, asubstituted lower alkyl group, a substituted alkaryl group, asubstituted aralkyl group, —Cl, —Br, —CN, —CF₃₁, —OCF₃, an -O-alkylgroup, an —S-alkyl group, an N-alkyl group, an O-alkenyl group, anS-alkenyl group, an N-alkenyl group, —SOCH₃, —SO₂CH₃, —ONO₂, —NO₂, —N₃,—NH₂, a heterocycloalkyl group, a heterocycloalkaryl group, anaminoalkylamino group, a polyalkylamino group, a substituted silylgroup, an RNA cleaving group, a reporter group, a DNA intercalatinggroup, a group for improving the pharmacokinetic properties of anoligonucleotide, a group for improving the pharmacodynamic properties ofan oligonucleotide, a methoxyethoxy group and a methoxy group.
 19. Theantisense oligonucleotide of claim 12, wherein at least one of saidnucleotides has a modified nucleobase.
 20. The antisense oligonucleotideof claim 19, wherein said modified nucleobase is selected from the groupconsisting of hypoxanthine, 5-methylcytosine, 5-hydroxymethylcytosine,glycosyl 5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,N⁶-(6-aminohexyl)adenine, 8-azaguanine, 7-5 deazaguanine and2,6-diaminopurine.
 21. The antisense oligonucleotide of claim 12,wherein said c-Jun protein is human c-Jun.
 22. A pharmaceuticalcomposition comprising the antisense oligonucleotide of claim 12, and apharmaceutically acceptable carrier.
 23. A pharmaceutical compositioncomprising: (a) a chemotherapeutic agent; (b) the antisenseoligonucleotide of claim 1, and (c) a pharmaceutically acceptablecarrier.
 24. A pharmaceutical composition comprising: (a) achemotherapeutic agent; (b) the antisense oligonucleotide of claim 12,and (c) a pharmaceutically acceptable carrier.
 25. A pharmaceuticalcomposition comprising: (a) an antisense oligonucleotide of claim 13;(b) an antisense oligonucleotide comprising 8 to 30 nucleotidesconnected by covalent linkages, wherein said antisense oligonucleotidehas a sequence specifically hybridizable with a nucleic acid encoding ac-Jun protein, wherein said antisense oligonucleotide inhibits theexpression of said c-Jun protein and wherein said antisenseoligonucleotide comprises at least one 2′-methoxyethoxy residue; and (c)a pharmaceutically acceptable carrier.
 26. A pharmaceutical compositioncomprising: (a) a chemotherapeutic agent; (b) an antisenseoligonucleotide of claim 13; (c) an antisense oligonucleotide comprising8 to 30 nucleotides connected by covalent linkages, wherein saidantisense oligonucleotide has a sequence specifically hybridizable witha nucleic acid encoding a c-Jun protein, wherein said antisenseoligonucleotide inhibits the expression of said c-Jun protein andwherein said antisense oligonucleotide comprises at least one2′-methoxyethoxy residue; and (d) a pharmaceutically acceptable carrier.27. A method of inhibiting the expression of a c-Fos protein in cells ortissues comprising contacting said cells or tissues with an antisenseoligonucleotide of claim 1, so that expression of said c-Fos protein isinhibited.
 28. A method of inhibiting the expression of a c-Jun proteinin cells or tissues comprising contacting said cells or tissues with anantisense oligonucleotide of claim 12, so that expression of said c-Junprotein is inhibited.
 29. An antisense oligonucleotide comprising 8-30nucleotides connected by covalent linkages, wherein said antisenseoligonucleotide comprises at least an 8 nucleobase portion of SEQ ID NO:10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 120,121, 122, 123, 131, 132, 133, 134, 135, 136, 137, or 138, wherein saidantisense oligonucleotide inhibits the expression of a c-Fos protein,and wherein said antisense oligonucleotide comprises two or morechemically distinct regions.
 30. The antisense oligonucleotide of claim29 wherein said antisense oligonucleotide is a hemimer or a gapmer. 31.The antisense oligonucleotide of claim 29 wherein one of said chemicallydistinct regions comprises one or more 2′-methoxyethoxy residues.
 32. Anantisense oligonucleotide comprising 8-30 nucleotides connected bycovalent linkages, wherein said antisense oligonucleotide comprises atleast an 8 nucleobase portion of SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 36,or 37, wherein said antisense oligonucleotide inhibits the expression ofa c-Jun protein, and wherein said antisense oligonucleotide comprisestwo or more chemically distinct regions.
 33. The antisenseoligonucleotide of claim 32, wherein said antisense oligonucleotide is ahemimer or a gapmer.
 34. The antisense oligonucleotide of claim 32,wherein one of said chemically distinct regions comprises one or more2′-methoxyethoxy residues.